0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response

Abstract

Purpose: To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis.

Methods: The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid–Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting.

Result: In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1 minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid–Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1β.

Conclusions: According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1β expression; and provided a potential treatment for fungal keratitis.

Translational Relevance: This study provides a potential treatment for fungal keratitis in the clinic.

Introduction

Fungal keratitis (FK) is an infectious corneal disease with a long course and poor prognosis that severely impairs vision. Ocular injury, excessive contact lens use, prolonged corticosteroid therapy, and postoperative corneal infection are the most common causes of fungal ocular infection.^1–3 In developing countries, Aspergillus fumigatus is one of the most common causes of FK.^4–7 Clinically, the first line of treatment for FK is a polyene antifungal antibiotic. Natamycin (NTM) is the only drug approved by the US Food and Drug Administration to treat FK,⁸ but it is not always effective for severe corneal fungal disease.^9,10 Other technologies being developed include new liposome and vesicle administration systems,¹¹ nano-carrying drugs^12,13 and hydrogel-carrying drugs.¹⁴ The prognosis is frequently poor owing to a scarcity of effective antifungal medications, fungus resistance, and poor penetration.^1,15–18 Therefore, it is critical to develop new anti-FK agents. 

Hypochlorous acid (HCLO) is weak acid with no charge that has high oxidative potency, physiological equilibrium solution stability,¹⁹ and high activity against bacteria, viruses, and fungal micro-organisms.²⁰ During neutrophil activation, respiratory bursts generate hydrogen peroxide and the activated granule enzyme myeloperoxidase (MPO) converts hydrogen peroxide to HCLO in the presence of Cl⁻ and H⁺.²¹ It is a natural molecule produced by neutrophils, with no evidence of microbial resistance.²² Clinically isolated Candida and Aspergillus have been shown to produce biofilms,^23,24 and HCLO has been shown to disrupt biofilm formation in nonocular or systemic studies.^25–30 

In clinical practice, 0.01% HCLO is used primarily to treat burn infections, diabetic foot ulcers, necrotizing fasciitis, stress ulcers, and atopic dermatitis. It is also an anti-itching and anti-inflammatory agent.^20,29,31 According to Hatanaka et al.,³² high-purity hypochlorite solutions inactivate coronaviruses with logarithmic 50% tissue culture infectious doses in 10 seconds and HCLO can be used as disinfectants for novel coronaviruses. Externalized HCLO has been shown by Jandova et al.³³ to inhibit skin inflammation gene expression and tumor progression in SKH-1 high-risk mice. The nasal and cranial cavities can also be rinsed with 0.01% HCLO.³⁴ Blepharitis and dysfunctional dry eyes can both be treated with 0.01% HCLO, according to research by Li et al.³⁵ According to the finding, the most effective effector in killing A. fumigatus mycelia is superoxide produced by neutrophil nicotinamide adenine dinucleotide phosphate oxidase.³⁶ In addition, Odorcic et al.³⁷ demonstrated that 0.01% HCLO decrease the number of yeast cells or mold conidia by 99.99% in 60 seconds and killed Candida albicans in 0 minutes.³⁸ 

In this study, we demonstrated the antifungal and anti-inflammatory effects of 0.01% HCLO in vitro and in vivo. Our study may lead to a new treatment option for FK. 

Materials and Methods

Culture and Preparation of A. fumigatus Conidia

The standard A. fumigatus lyophilized powder (ATCC96918) was purchased from BeNa Culture Collection (Kunshan City, China). The strain was activated in potato dextrose agar (PDA) for 5 days at 28°C. Collected spores with sterile water and adjusted to a concentration referred to the method of Balouiri et al.³⁹ 

Time Kill Assay

To assess the bactericidal effect, time kill assays were carried out. The spore concertation was adjusted to 1.0 × 10⁶ colony-forming units (CFU)/mL. We added 100 µL spore suspension to 900 µL 0.01% HCLO and mixed for 1, 5, and 15 minutes. Then we transferred 100 µL of the mixture into 900 µL DE neutralizing broth (Qingdao, Shandong, China) for 5 minutes to stop antimicrobial activity. We inoculated 100 µL in PDA medium for 48 hours at 35°C. The number of colonies was counted on a black background. 

Broth Microdilution Method

Broth microdilution methods were performed to test antimicrobial susceptibility, with suitable modification. The spore concentration was adjusted to 1.5 × 108 CFU/mL using Sabouraud liquid medium (SDB), and 100 µL of spore suspensions were added to wells of 96-well plates. Phosphate-buffered saline (PBS), 0.01% HCLO, and 5% NTM were added to each wall separately, with SDB spore suspension serving as blank controls. Absorbance (optical density value) at 540 nm was measured by an enzyme marker after 24 hours of incubation at 35°C. The supernatant was removed and added 50 µL of fungal fluorescent staining solution (Guokang, Shandong, China) to observe the fungal growth under a fluorescent microscope. 

Animal Models of FK

Healthy SD rats (male, 6–8 weeks old) were purchased from the Animal Experiment Center of Xuzhou Medical University (Xuzhou, Jiangsu, China) and treated according to the ARVO Statement for Animals for Ophthalmic and Vision Research. The right eye of the rats was the experimental eye and the left eye was blank for control. Rats were anesthetized with 2% pentobarbital. A trephine was used to outline a 3.5-mm circle on the cornea under a stereomicroscope, scraped off the scope corneal epithelium. We used a 30G needle to scratch the superficial stroma, take 10 µL disposable inoculation loop to pick off A. fumigatus spore colonies to cover the corneal surface, and then covered soft contact lens and sutured the eyelids. The sutures were removed 48 hours later. 

Animal Groupings and Treatments

Rat models of A. fumigatus keratitis were divided into three groups of six rats each. PBS, 0.01% HCLO, and 5% NTM were dropped into the right eye of each group once an hour for 10 hours. The corneal response and corneal fluorescein staining in rats were examined and photographed on days 1, 3, and 5 after treatment using a slit-lamp microscope. In vivo confocal microscopy was performed on the same day. The rats were sacrificed at days 1, 3, and 5 postinfection (p.i.), and the corneal were harvested for tissue homogenate plate count and histological examination. 

Clinical Score

Clinical scoring was performed under a slit-lamp microscope, and all clinical scorers were unaware of the experimental design. According to the evaluation system of Wu et al.,⁴⁰ the score was divided into three criteria: ulcer area percentage, lesion depth, and surface regularity; each criterion was divided into levels of 0 to 4, with 1 point added to each level. The sum of the three scores was scored in total, with scores of 1 to 4 being mild, 5 to 8 being moderate, and 9 to 12 being severe. 

In Vivo Confocal Microscopy

After adequate anesthesia, we fixed the rats and advanced the cornea module of the confocal microscope until the gel contacted the central surface of the cornea. The depth and position of the focus were adapted, and images of the corneal ulcer and the surrounding cornea were acquired. Mycelial density and inflammatory cell measurements were performed using ImageJ software. 

Plate Count

To evaluate the corneal fungal load of rats in different treatment groups (n = 3/group) on day 3, corneal homogenate, gradient dilution, and cultured in PDA media at 35°C for 48 hours. The colonies were photographed and counted and expressed as CFU per cornea. 

Periodic Acid–Schiff (PAS) Staining

Eyeballs of rats were fixed in 4% paraformaldehyde for 48 hours at 4°C, the lens was removed, paraffin embedded, cut into 5-µm sections, mounted on slides, and stained with PAS staining. Paraffin sections were dewaxed and rehydrated, with periodic acid for 6 minutes and Schiff's reagent for 20 minutes, and rinsed under running water. Fungal load and morphological features in corneal tissue were observed under the light microscope. 

Fungal Fluorescent Staining

Fungal fluorescence staining is a method of detecting fungi that combines calcofluor white with chitin components of the fungal cell wall to produce blue fluorescence under ultraviolet excitation light. Experiments have shown that fungus fluorescence staining is more sensitive than PAS staining.^41,42 The paraffin sections were dewaxed and rehydrated through to water, stained according to the protocol of the fungal fluorescent staining solution, had the fluorescent staining solution added dropwise for 1 minute, and sealed. We observed the fungal load and morphological characteristics of corneal tissue under a fluorescence microscope at 320 to 340 nm UV light. 

MPO Assay

The corneas of the treatment group were collected and homogenized, the MPO was measured by 460-nm spectrophotometry with an MPO detection kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) at 37°C. The slope of the line is related to the MPO unit per gram of the cornea. 

Immunofluorescence Staining

Eyeballs were embedded into optimum cutting temperature compound and 5-µm cryosections were obtained. After washing, the plates were blocked with 3% bovine serum albumin for 1 hour at room temperature. The anti-rat neutrophil monoclonal antibody (1:1000, Fitzgerald Industries, Hampton, NH) was applied sequentially with overnight incubations at 4°C, and then incubated with fluorescein isothiocyanate–labeled goat anti-rabbit secondary antibody (1:500; Abbkine, Wuhan, China) for 1 hour at room temperature, and the nuclei were stained with 4′,6-diamidino-2-phenylindole dibasic acid. After three washes in PBS, the portions were sealed with 50% glycerol. A fluorescence microscope was used to take pictures (Leica Microsystems, Wetzlar, Germany) (original magnification ×200). 

Western Blot

Cornea tissues were collected and homogenized in RIPA (Beyotime, Shanghai, China) buffer with PMSF (Beyotime, Shanghai, China) (RIPA: PMSF = 100:1), and lysed on ice for 30 minutes. The supernatant was removed after centrifugation at 4°C (15 minutes as 12,000 rpm) and protein quantification was performed by BCA protein assay (ElabScience, Wuhan, China). Then transferred to an 0.22-µm polyvinylidene difluoride membrane (BioSharp, Tallinn, Harjumaa, Estonia) after separation by 12.5% sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The membranes were blocked with 1% skim milk powder for 1 hour, the primary antibody against β-actin (1:5000 BioWorld, Nanjing, China), tumor necrosis factor (TNF)-α (1:1000; Abcam, Branford, CT) and IL-1β (1:1000 BioWorld) were applied sequentially with overnight incubations at 4°C, and the membranes were washed three times in Tris-buffered saline with Tween. The secondary antibody was incubated for 1 hour at room temperature, washed three times with -buffered saline with Tween, and blotted with chemiluminescence (ECL; Thermo Fisher Scientific, Waltham MA) for chemiluminescence observation. 

Statistical Analyses

A t-test was used to compare the differences between the two groups. Statistical differences between three or more groups were analyzed by one-way analysis of variance and by performing multiple comparisons of multiple groups. A P value of less than 0.05 was considered significant. Graphs were plotted and statistically analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Immunofluorescence staining and protein blotting were analyzed using ImageJ (National Institutes of Health, Bethesda, MD). 

Result

0.01% HCLO Can Kill A. fumigatus Spores Quickly

Time kill assay showed that, after 0.01% HCLO treatment of A. fumigatus spores for 1, 5, and 15 minutes, no significant colony growth on PDA after 48 hours of incubation. The PBS groups had a large amount of fungal growth (Fig. 1A). The results obtained with the broth microdilution test are shown in Figure 1B. After 24 hours of culture, the absorbance value in the 0.01% HCLO and 5% NTM groups were significantly lower than SDB and PBS groups (Fig. 1B) (P < 0.01). No statistically significant differences were detectable between the HCLO and NTM groups (Fig. 1B). No significant mycelial growth was observed in fluorescence microscope observations in HCLO and NTM groups (Fig. 1C). 

Figure 1.

 

(A) Mixed 1-, 5-, and 15-minute and cultured, group HCLO no colony growth. (B) After incubation for 24 hours, optical density value at 540 nm showed that the 0.01% HCLO-treated group was significantly lower than PBS group (*P < 0.05, **P < 0.01). (C) Staining of the mycelium indicated that 0.01% HCLO almost completely inhibited A. fumigatus growth (10×).

Figure 2.

 

(A) Clinical scores at 1, 3, and 5 days p.i. (B) Representative slit lamp photographs of PBS-, 0.01% HCLO-, and NTM-treated rat cornea. (C) In vivo confocal microscopy images of mycelial at 3 days p.i. and percentage of mycelium density (D). (E, F) In vivo confocal microscopy images of inflammatory cells at 3 days p.i. and quantitative analysis (F). **P < 0.01; ***P < 0.001; ^#P < 0.05.

Figure 2.

 

Continued.

Therapeutic Treatment With 0.01% HCLO

Clinical scores were increased significantly at 3 and 5 days p.i. in the PBS-treated group, and were decreased markedly in favor of the group treated with HCLO on days 3 and 5 after the start of treatment (Fig. 2A). The clinical scores of the HCLO and NTM treatment groups were lower than PBS treatment group at 3 and 5 days p.i. (P < 0.001). There was no statistically significant difference in clinical scores between the HCLO and NTM groups (Fig. 2A). Slit-lamp examination showed most of the PBS-treated corneas were perforated at 5 days p.i.. However, at 3 and 5 days p.i., the size of the ulcer of HCLO-treated and NTM-treated groups decreased and localized, and the cornea was more transparent than PBS-treated group (Fig. 2B). Thus, 0.01% HCLO would decrease the clinical score and the cornea was more transparent. In vivo confocal microscope images showed that the mycelia fracture was twisted and shorter in HCLO-treated and NTM-treated groups. Although the mycelia in the PBS-treated group were thick and vigorous at 3 days p.i. (Figs. 2C and 2D), compared with the PBS-treated group, the mycelium density was decreased in the HCLO-treated and NTM-treated groups (P < 0.01). The number of inflammatory cells in the HCLO group and NTM group decreased compared to the PBS treatment group (P < 0.01), and the HCLO treatment group showed a significant decrease in the number of inflammatory cells compared to the NTM treatment group (P < 0.05) at 3 days p.i. (Fig. 2F). The results of fluorescence staining of fungi and PAS staining showed that fewer fungi were observed in group HCLO and group NTM, and the hyphal breakage became shorter (Fig. 3A through 3C). The fungal load in HCLO-treated and NTM-treated groups was significantly lower than in PBS group at 3 days p.i. (P < 0.05) (Figs. 3D and 3E) (P < 0.001). These results show that 0.01% HCLO can decrease the fungal load, inhibit hyphal growth, and decrease histopathological damage. 

Figure 3.

 

(A) PAS staining of corneal tissue sections of three groups at 3 days p.i. (original magnification ×200). (B) Fungal fluorescent staining of corneal tissue and quantitative analysis (C) at 3 days p.i. (original magnification ×200). (D, E) Representative plates count of PBS-, 0.01% HCLO-, and NTM-treated groups (D) and quantitative diagram (E). ***P < 0.001. ns, not significant.

0.01% HCLO Decreases the Number and Vitality of Neutrophils in A. fumigatus Keratitis Rat Model

Immunofluorescence staining was used to calculate the fluorescence intensity of neutrophils in the corneas of the rats treated with PBS or NTM and HCLO, showing that HCLO treatment significantly decreased the infiltration of neutrophils compared with PBS and NTM treatment (Fig. 4A). The fluorescence intensity of neutrophils between the HCLO-treated group and the PBS and NTM group was statistically significant (Fig. 4B) (P < 0.01). Moreover, MPO results suggested that HCLO treatment inhibited neutrophil vitality in A. fumigatus keratitis rat model (Fig. 4C), and the inhibitory effect was more significant than that of NTM treatment (P < 0.001). 

Figure 4.

 

(A) Representative immunofluorescence staining images of rat corneas (A) and quantitative analysis (B). Green: PMN; Blue: nuclear staining 4′,6-diamidino-2-phenylindole dibasic acid; original magnification ×200. (C) Quantitative analysis of MPO at 3 days p.i. **P < 0.01; ***P < 0.001; ^###P < 0.001.

0.01% HCLO Decreases the Inflammatory Response in an A. fumigatus Keratitis Rat Model

To detect the effects of 0.01% HCLO in the inflammatory response, protein expression of different inflammatory factors was evaluated at 1, 3. and 5 days after infection. The protein expression levels of IL-1 and TNF-α in the HCLO-treated group were significantly lower than those in PBS-treated group at days 3 and 5 p.i. (Figs. 5A and 5C) (P < 0.001), with the most obvious difference on day 3. The difference was statistically significant (Figs. 5B and 5D) (P < 0.001), indicating that 0.01% HCLO could inhibit the expression of inflammatory factors IL-1 and TNF-α in rat model of A. fumigatus keratitis. 

Figure 5.

 

(A–D) Western blot results (A, B) and grayscale analysis (C, D) of TNF-α and IL-1β. ***P<0.001.

Discussion

FK is a challenging corneal infection, and FK is more severe than bacterial keratitis owing to the invasiveness and toxicity of fungi.⁴³ Currently, corneal ulceration, perforation, or even loss of the eye occur frequently during the disease process owing to a lack of effective antifungal agents and an overactive innate immune response.⁴⁴ FK has a long course in humans. Neutrophils are the earliest cells of the body's immune response and are an essential component in the fight against pathogens, but an excessive aggregation of neutrophils leads to an excessive inflammatory response, convening the presence of various immune cells and inflammatory factors, resulting in corneal damage.^45–47 Therefore, inhibiting fungi and excessive inflammation is equally important in the treatment of FK. In our study, we investigated the spore-killing effect of 0.01% HCLO in vitro and the antifungal and anti-inflammatory effects in a rat model. 

As a natural immune molecule, activated neutrophils produce HCLO as part of the body's innate immunity to invading pathogens.²² HCLO, a low-molecular-weight weak acid with no charge, is highly effective against all human pathogens, including bacteria, viruses, and fungi.⁴⁸ Fungal biofilms are composed of matrix-enclosed microbial populations that adhere to each other and to surfaces, forming organized communities. The structural barrier of this extracellular matrix makes these organisms less susceptible to antibiotic and antifungal treatments.⁴⁹ Low concentrations of HCLO can kill fungi rapidly and disrupt the formation of fungal biofilms.³⁰ Sakarya et al.²⁹ have shown that HCLO can actively penetrate biofilms and kill micro-organisms within them. Our data indicated that 0.01% HCLO can kill A. fumigatus spores within 1 minute, which is consistent with report from Odorcic et al.³⁷ In addition, we observed a significant decrease in optical density in the HCLO group, and no obvious fungal growth was observed in the hyphal fluorescence staining. In contrast, the growth of Aspergillus in the PBS group was cross-linked, indicating that 0.01% HCLO has a significant killing effect on Aspergillus growth and may have a destructive effect on fungal biofilms. 

In the treatment of FK, the therapeutic effect of drugs is related to their corneal penetration. However, 5% NTM is a suspension dosage form with poor water solubility and limited corneal penetration. In our study, we found that 0.01% HCLO decreased the area of corneal ulceration and the degree of corneal edema, shortened the course of the disease, and significantly decreased the number of viable fungal colonies compared with the PBS treatment group at 3 days p.i., as demonstrated by plate count. This finding is consistent with the results of PAS and in vivo confocal microscopy, which showed that HCLO significantly decreased the fungal load at the infection site. Our findings suggest that HCLO can limit the growth of Aspergillus and improve FK. This report is the first time that the antifungal effect of HCLO has been verified in the FK rat model. The therapeutic effect of HCLO may be related to its small molecular size, lack of charge, and water solubility, which may allow for better corneal penetration compared with NTM. However, further research is needed to confirm this hypothesis. 

Innate immunity is the first line of defense against fungal infection, and excessive inflammatory factors lead to corneal damage and a poor prognosis for FK.^45,50 Therefore, drugs that have both anti-inflammatory and antifungal effects are a good choice for treating FK. Experiments showed that HCLO promotes wound healing, significantly reduces inflammation signs,⁵¹ and decreased histamine, leukotriene B4, IL-2, IL-6, and IL-19 levels.^20,33 In rats with A. fumigatus keratitis in our study, we found that 0.01% HCLO decreased the number of inflammatory cells in the cornea, attenuated neutrophil activity and infiltration, and suppressed the protein expression levels of the proinflammatory cytokines TNF-α and IL-1β in the cornea. HCLO has the dual effect of killing A. fumigatus and inhibiting excessive inflammation. In our study, 0.01% HCLO decreased clinical scores, fungal load, or density of hyphae in the cornea, resulting in a similar clinical outcome as NTM treatment. However, the HCLO group observed more transparent corneal tissue under a slit-lamp microscope. Furthermore, the number of inflammatory cells, MPO activity, and neutrophil infiltration in the cornea were significantly lower after HCLO treatment than after NTM treatment at 3 days p.i. The anti-inflammatory effect of HCLO has been confirmed, but its underlying mechanisms require further investigation. In the treatment of FK, the long-term use of NTM may cause side effects such as eye redness, foreign body sensation, and drug particle adhesion to the eyes. However, HCLO has a rapid neutralization property and does not have eye surface toxicity.³⁰ Animal experiments have shown that topical eye drops with 0.01%, 0.03%, and 0.1% of HCLO aqueous solution did not show eye irritation symptoms.²² Although no significant ocular damage was observed in the experiment, further research is needed to demonstrate the ocular safety and efficacy of HCLO. 

Conclusions

HCLO has demonstrated the ability to kill Aspergillus in vitro and has shown promising therapeutic effects in a rat model of Aspergillus keratitis. It may have potential as a clinical treatment for FK. 

Acknowledgments

Disclosure: K. Zhao, None; F. Hu, None; Z. Zhang, None; X. Yin, None; H. Wang, None; M. Li, None