BITC stock solution (100 mg/ml) was prepared by dissolving BITC (MEC, Shanghai, China) in dimethyl sulfoxide (DMSO, Solarbio, Beijing, China). BITC was further diluted to the concentrations of 50, 100 and 200 μg/ml for calcofluor white (CFW) staining, time-kill assay, propidium iodide (PI) uptake assay, reactive oxygen species (ROS) assay, fungal adhesion assay, and crystal violet assay. A. fumigatus mice model was treated with 200 μg/ml BITC, and RAW264.7 cells were treated with 3 and 6 μg/ml BITC.

RAW264.7 cells (from Shanghai Chinese Academy of Sciences, China) were incubated in high glucose DMEM with 10% FBS at 37°C with 5% CO₂. Human corneal epithelial cells (HCECs, from the Laboratory of the University of Xiamen, Fujian, China) were cultured in DMEM supplemented with an equal volume of Hams F12 and 5% FBS at 37°C with 5%CO₂. RAW264.7 cells or HCECs were seeded in 96-well plates. Once cells reached 80% confluence, 100 μl of BITC (0, 3, 6, 12, 25, 50, 100, 200, 400, and 800 μg/ml) or 0.1% DMSO was added to each well and cultured for 24 h. Cell Counting Kit-8 (CCK-8; MCE) (10 μl) was added to each well, then absorbance (450 nm) was measured.

The experimental method is derived from earlier studies (Zhu et al., 2021). Briefly, normal mice were given 5 μl of BITC (100, 200, 400, and 800 μg/ml) into the conjunctival sac four times daily. Adverse effects of BITC on the cornea were evaluated by corneal fluorescein staining (CFS) score at 0, 1, 3, and 5 days. CFS scoring criteria refer to the Organization for Economic and Cooperative Development (OECD) grading scale for ocular irritation, which comprised corneal opacification density and area, iritis severity, conjunctival redness, edema, and secretion.

Aspergillus fumigatus strain (NO3.0772) was purchased from China General Microbiological Culture Collection Center. A. fumigatus conidia and hyphae were prepared using the previous technique (Tian et al., 2021). Conidia suspension (1 × 10⁵ CFU/ml) was used in minimum inhibitory concentration (MIC) assay, CFW staining, time-kill assay, PI uptake assay, biofilm inhibition assay, ROS assay, SEM, TEM, and fungal adhesion assay. Activated hyphae (3 × 10⁸ CFU/ml) were used to infect the cornea of mice model, and inactivated hyphae (5 × 10⁶ CFU/ml) were employed in cell experiments.

The antifungal capacity of BITC at different concentrations was tested by MIC and CFW. Conidia were seeded in a 96-well plate and subjected to BITC (0, 25, 50, 100, 200, 400, and 800 μg/ml) for 24 h. The optical density (OD) at 570 nm was measured. CFW staining was aimed to detect the antifungal of BITC to hyphae. CFW (Sigma, MO, United States) is a specific fluorescent dye for fungal cell wall chitin that binds to live fungi (Green et al., 2000). Hyphae in 6-well plates were cultured with BITC at 28°C for 6 h, the supernatant was then withdrawn and 1 ml CFW was added. A fluorescence microscope (Nikon, Tokyo, Japan, ×200) was used to capture the image.

Fungicidal/fungistatic activities were evaluated by time-kill assay. The conidial suspension was incubated with 0.1% DMSO or BITC at 37°C, 120 rpm. The mixture (100 μl) from different times (0, 4, 6, 8, 12, and 24 h) of BITC treated conidial suspension was plated on Sabouraud agar plates and incubated at 37°C for 24 h, respectively. The colony-forming units (CFU) were counted. When compared to CFU on the mediums at 0 h, a reduction in colony count >2log10 CFU/mL was defined as fungicidal activity, and in colony count <2log10 CFU/mL was defined as fungistatic activity (Balouiri et al., 2015).

To determine the integrity of the fungal membrane, PI uptake assays were used after treatment with 0.1% DMSO or BITC. Experimental procedures refer to the previous method (Tian et al., 2021).

Morphological and organelle changes in fungal conidia and hyphae were determined by SEM and TEM. Hyphae were germinated by A. fumigatus conidia at 37°C for 24 h, and then treated with 0.1% DMSO or BITC (200 μg/ml) for 8 h in 24-well plates. According to the previous method, hyphae were collected, immobilized, and dehydrated after three PBS rinses (Tian et al., 2021). Images were observed with SEM (JSM-840; JOEL Company, Japan, magnification ×2,000 and ×5,000) and TEM, respectively, (JEM-1200EX; JOEL Company, Japan, magnification ×15,000 and ×40,000).

ROS content in A. fumigatus conidia was tested by the ROS fluorometric assay kit (Elabscience, E-BC-K138-F). 10 μM 2′,7′-dichlorofluorescien diacetate (DCFH-DA) was added into conidia suspension which was treated with BITC for 6 h at 28°C before to label intracellular ROS. Conidia suspension treated by H₂O₂ for 2 h was regarded as the positive control. Fluorescence intensity was measured at an excitation wavelength of 500 nm and an emission wavelength of 525 nm.

HCECs were inoculated and cultured on chambered slides (4/slide) for 24 h at 37°C. Then, BITC and A. fumigatus conidia were added and incubated for 4 h. PBS rinse was performed three times to get rid of non-adherent conidia. Hematoxylin–eosin (HE) staining which refer to previous articles (Jia et al., 2022) was applied to the slides. Finally, we observed and photographed specimens by microscopy (Nikon, Tokyo, Japan, ×400), and conidia adherence to HCECs was counted.

Conidia were incubated in 24-well plates at 37°C for 48 h. Then, 500 μl of BITC was applied for 24 h. Each well was stained with 0.1% crystal violet (CV; Solarbio, Beijing, China) for 15 min and decolorized with 95% ethanol for 5 min. Finally, a new 96-well plate was used to transfer the supernatant and the OD value (570 nm) was measured.

Anti-inflammatory and antifungal effects of BITC in vivo were evaluated using C57BL/6 mice (female, 7–8 weeks old, SPF; Pengyue Co. Ltd. Jinan, China). The modeling procedures refer to the previous method (Zhu et al., 2021). Briefly, A. fumigatus hyphae (5 μl; 3 × 10⁸ CFU/ml) were applied topically to the wounded corneal epithelium of anesthetized mice of left eye, a soft contact lens was placed, and eyelids were sutured. After 24 h, the eyelids sutures were removed. Then, left eyes of mice were injected subconjunctivally with 5 μl of BITC (200 μg/ml) or 5 μl of natamycin (NATA, CAS 7681-93-8; Macklin Biochemical Co. Ltd., Shanghai, China, 5 μg/ml) per day. Infected mice who administered DMSO (0.1%) in the same volume were regarded as control group. A slit-lamp microscope and clinical score (n = 5/group/time) were used to evaluate the severity A. fumigatus keratitis at 1, 3, and 5 days post infection (p.i.) (Zhu et al., 2021). Pathology of infected corneas (n = 6/group/time) was presented by HE staining at 3 days p.i. The work was authorized by the Research Ethics Committee of the Affiliated Hospital of Qingdao University, and the treatments given to the mice confirm to the ARVO Statement regarding the Use of Animals in Ophthalmology and Vision Research.

Infected corneas from 3 days p.i. were added to sterile PBS (0.1 ml) and ground into homogenate (n = 4/group). Sabouraud agar plates were used to incubate fungal colonies in corneal homogenates at 37°C for 24 h. The CFU on the plates was photographed and counted.

The amount of polymorphonuclear neutrophilic leukocytes (PMN) was quantitated by MPO kit (Njjcbio, Jiangsu, China). Corneas of FK mice (n = 6/group) at 3 days p.i. that treated by BITC or DMSO were tested the level of MPO in accordance with the guidelines. An enzyme-labeling instrument was used to measure absorbance (460 nm).

RAW264.7 cells cultured as mentioned above were pretreated with or without BITC (3 μg/ml) for 2 h, and subsequently stimulated with inactivated A. fumigatus hyphae for 8 h or 24 h as required. Mincle ligand trehalose-6,6-dibehenate (20 μg/ml) (TDB Invivogen, San Diego, CA, USA) was also used to stimulate RAW264.7 cells which were pretreated with BITC for 2 h.

Infected and uninfected mice corneas (n = 6/group/time) were added to RNAiso Plus reagent (Takara, Dalian, China) and homogenized by the TissueLyser II (28 Hz, 20 min; QIAGEN) at 3 and 5 days p.i. (Yin et al., 2022). The mRNA levels of tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, and Mincle were detected in corneas supernatant. Likewise, the mRNA levels of RAW264.7 cells at 8 h after A. fumigatus or TDB stimulation were tests. RNAiso Plus reagent was used to extract total RNA. After spectrophotometric quantification (260 nm), the reverse transcription kit (Vazyme, Nanjing, China) synthesized the total mRNA into cDNA. RT-PCR reaction steps have been described in previous studies (Zhu et al., 2021). Table 1 displays the RT-PCR primer sequences.

Mice corneas (n = 5/group/time) for 3 and 5 days p.i. and RAW264.7 cells after stimulation of inactivated hyphae or TDB for 24 h were collected for detection of Mincle protein levels, which were fully lysed in RIPA solution (Solarbio, Beijing, China) with 1% PMSF and Phosphatase inhibitor. Concentrations of protein were then determined by a BCA Assay Kit (Elabscience). Separated proteins in PVDF (polyvinylidene fluoride) membranes (Solarbio) were transferred from SDS-PAGE glue. After 2 h’s blocking with a blocking buffer (Beyotime Biotechnology, Shanghai, China), the PVDF membranes containing proteins were incubated with goat anti-mouse Mincle (1:500, Santa Cruz, CA, United States) antibody or goat anti-rabbit β-actin (1:2000, Elabscience) antibody overnight at 4°C and with the corresponding secondary antibody (1:2000, Elabscience) for 2 h at 28°C. Chemiluminescence (ECL; Thermo Fisher Scientific, United States) was used to inspect the blots.

Corneas (n = 5/group/time) were collected, homogenized, and centrifuged (12,000 rpm, 5 min) according to the reported literature at 3 and 5 days p.i. (Yin et al., 2022). RAW264.7 cell cultures supernatants (n = 6/group) were harvested at 24 h after stimulation. Protein expression of TNF-α and IL-1β was measured by ELISA kits (R&D system, United States). Experimental procedures were performed according to the manufacturer’s instructions. The absorbance (450 nm and 570 nm) was measured.

RAW264.7 cells were seeded and treated by BITC for 24 h in chambered slides (4/slide) plates. After fixation with paraformaldehyde, chambered slides (4/slide) containing cells were incubated overnight with Mincle (1:200; Santa Cruz, CA, United States) primary antibody and FITC-conjugated IgG goat anti-rat secondary antibody (1:200; Elabscience) for 1 h, and DAPI (ready-to-use, Solarbio) to label the cell nucleus. Finally, fluorescent microscopy (Zeiss Axio Vert; magnification ×400) was used to observe and take pictures, and the OD in the pictures was analyzed by Image J software.

Data were presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8 software. The Mann–Whitney U test examined differences in clinical scores. Unpaired two-tailed Student’s t-test was used to analyze the data from MPO assay. Data from CCK-8 assay, MIC assay, time-kill assay, biofilm assay, RT-PCR, Western blot, ELISA, plate count, and IFS were analyzed by one-way analysis of variance (ANOVA) test. Further two-by-two comparison was performed using Bonferroni analysis. p < 0.05 was considered difference significant. Three times each experiment was repeated.

To explore the toxicity of BITC and the optimal concentrations for subsequent research on HCECs and RAW264.7 cells, we performed CCK-8 experiments with a range of concentrations of BITC (0, 3, 6, 12, 25, 50, 100, 200, 400, and 800 μg/ml). Compared with control groups, the absorbance remained the same in HCECs (Figure 1A) after treated with 100 μg/ml BITC, and in RAW264.7 cells (Figure 1B) after treated with 6 μg/ml BITC for 24 h, indicating the above-mentioned concentration of BITC is safe for HCECs and RAW264.7 cells. Next, we evaluated the performance of BITC on mice corneas by the Draize eye test. No anterior segment damage (conjunctiva, cornea, anterior chamber, and iris) and corneal staining tested by fluorescein sodium were discovered after BITC treatment (100–400 μg/ml) for 1, 3, and 5 days, as well as after 800 μg/ml BITC treatment for 1 day (Figures 1C,D). Despite the fact that conjunctival congestion and corneal neovascularization were not found, CFS displayed a small amount of punctate staining of the cornea after receiving BITC treatment at 800 μg/ml for 3 and 5 days.

The antifungal effect of BITC at different concentrations was evaluated during the growth of A. fumigatus. MIC results showed that the growth and germination of A. fumigatus were both inhibited by treatment of BITC (25 μg/ml) for 24 h (p < 0.05). As concentration of BITC raised, the inhibition rate increased until it reached 100% at 200 μg/ml of BITC (Figure 2A). Data from the time-kill assay demonstrated that the amount of A. fumigatus CFU was significantly decreased in BITC-treated groups at 50, 100, or 200 μg/ml compared to the control group at each time point (Figure 2B). As treatment time extended, the number of CFU at 50 μg/ml of BITC (red line) and 100 μg/ml of BITC (blue line) declined steadily in a concentration-dependent manner. While the number of the colonies was decreased rapidly by BITC (200 μg/ml; black line) at 3 h, and almost 0 at 18 h. Images of CFW showed intuitively the surviving hyphae (blue fluorescence; Figure 2C) after treatment of BITC at 50, 100, and 200 μg/ml. The hyphae treated with DMSO were more robust and dense, while BITC treatment significantly reduced the amount of surviving hyphae that were slender and short. The number of viable hyphae decreased with increasing BITC concentration.

PI staining showed negative fluorescence in A. fumigatus treated with 0.1% DMSO, whereas in A. fumigatus treated with BITC at concentrations of 50, 100, and 200 μg/ml, red fluorescence was observed (Figure 3A). Additionally, the fluorescence of PI was increased by BITC in a dose-dependent manner. In SEM images, there were no apparent morphological alterations in untreated hyphae, which were long and with smooth surface (Figures 3B,C). Contrarily, hyphae exposed to BITC (200 μg/ml) for 8 h exhibited shrinkage and deformation, and were broken into fragments (Figures 3D,E). TEM images (Figures 3F,G) revealed the cellular ultrastructure in DMSO-treated and BITC-treated conidia and hyphae of A. fumigatus. A normal cellular ultrastructure with homogenous cell wall thickness, cytoplasmic density, and normal mitochondrial morphology was observed in untreated conidia and hyphae of A. fumigatus. However, it could be seen detachment of cell membrane from the cell wall and loss of the integrity of cell membrane in both conidia and hyphae after BITC (200 μg/ml) treatment for 8 h. Additionally, conidia (Figure 3F) showed cell wall thickening, loose cytoplasmic and disrupted mitochondrial morphology, and hyphae (Figure 3G) displayed distorted and shriveled morphology and swollen mitochondria with destroyed cristae structures. The level of ROS in A. fumigatus conidia was measured by fluorescent probe DCFH-DA. As shown in Figure 3H, treatment of BITC (50, 100, and 200 μg/ml) for 6 h caused a sustained increase in the level of ROS. With the increase of BITC concentration, the accumulation of ROS in conidia was upregulated significantly (p < 0.05).

Aspergillus fumigatus conidia in the control group had strong adherence to HCECs, with about 5 conidia adhering to each HECE. Only a small number of conidia stuck to HCECs under treatment of BITC (50, 100, 200 μg/ml) after 4 h (Figures 4A,B). Seldom conidia adherence could be observed under the treatment of BITC (200 μg/ml). The inhibition of BITC on mature biofilms was quantified by measuring the absorbance of CV staining. Absorbances of stained biofilms were decreased significantly by BITC at 100 and 200 μg/ml (Figure 4C; p < 0.001).

BITC, NATA, or DMSO were applied topically in order to evaluate the protective effect in A. fumigatus keratitis mice of BITC. No differences were observed in corneal ulceration and opacity demonstrated by slit-lamp photography (Figure 5A) at 1 day p.i. While the 200 μg/ml BITC (p < 0.01, p < 0.001) and 5 μg/ml NATA groups (p < 0.0001, p < 0.0001) alleviated corneal ulceration and opacity (Figure 5A), and obtained lower clinical scores (Figure 5B) compared to the 0.1%DMSO group at 3 and 5 days p.i. In addition, plate counting (Figures 5C,D) experiments showed that both 200 μg/ml BITC (p < 0.001) and 5 μg/ml NATA (p < 0.001) considerably decreased fungal load in the infected corneas when compared to the DMSO-treated group and no difference between the BITC and NATA groups (p > 0.05).

Next, we investigated how BITC treatment affected corneal immune cell infiltration and expression of pro-inflammatory cytokines after infection. The expression of pro-inflammatory cytokines in mice corneas was tested by RT-PCR and ELISA. PCR data showed decreased IL-6 mRNA in the 200 μg/ml BITC-treated group than in DMSO-treated group at 3 days p.i. (Figure 6A; p < 0.05). Moreover, our results revealed that the mRNA expression of IL-1β (Figure 6B; p < 0.001, p < 0.5) and TNF-α (Figure 6C; p < 0.01, p < 0.01) was considerably decreased following BITC treatment compared with DMSO-treated infected corneas at 3 and 5 days p.i. Consistently, the protein expression of IL-1β (Figure 6D; p < 0.001, p < 0.01) and TNF-α (Figure 6E; p < 0.05, p < 0.001) in the 200 μg/ml BITC-treated group was lower than DMSO-treated group. MPO levels were lower in 200 μg/ml BITC-treated mice corneal than DMSO-treated group at 3 days p.i. (Figure 6F; p < 0.001). In addition, HE staining showed a large infiltration of immune cells in the corneal stroma of FK at 3 and 5 days p.i. (Figure 6G). While corneal infiltration of immune cells in the 200 μg/ml BITC and 5 μg/ml NATA treatment groups were alleviated at 3 and 5 days p.i. (Figure 6G). In infected corneas, pro-inflammatory cytokines were downregulated by BITC, and the effect of BITC in RAW264.7 cells stimulated by A. fumigatus was verified further. RT-PCR results manifested that the expression of IL-1β (Figure 6H; p < 0.0001, p < 0.0001), TNF-α (Figure 6I; p < 0.001, p < 0.001) and IL-6 (Figure 6J; p < 0.0001, p < 0.0001) mRNA was significantly reduced by BITC (3 and 6 μg/ml) after 8 h of A. fumigatus stimulation in a concentration-dependent manner. Protein levels of IL-1β (Figure 6K; p < 0.0001) and TNF-α (Figure 6L; p < 0.0001) were similarly noticeably reduced in the BITC-treated (3 μg/ml) group after 24 h.

The data from RT-PCR and western blot indicated that the mRNA and protein levels of Mincle were elevated notably after A. fumigatus infection in RAW264.7 cells (Figures 7A–C; p < 0.001, p < 0.0001), and Mincle expression reduced under the treatment of BITC (3 μg/ml and 6 μg/ml) (p < 0.001, p < 0.0001). The same result was observed in infected corneas that BITC (200 μg/ml) significantly downregulated the Mincle mRNA (Figure 7D; p < 0.001, p < 0.001) and protein (Figures 7E,F; p < 0.001, p < 0.0001) levels compared to the control group at 3 and 5 days p.i. RAW264.7 cells were stimulated by TDB, a Mincle specific agonist, to further define the role of BITC on Mincle. Our results revealed that treatment of BITC (3 μg/ml) suppressed the elevated expression of Mincle (Figures 7G–I; p < 0.0001, p < 0.001), IL-1β (Figures 7J,K; p < 0.0001, p < 0.0001), TNF-α (Figures 7L,M; p < 0.05, p < 0.05), and IL-6 (Figure 7N; p < 0.0001) at mRNA and protein levels that stimulated by TDB. IFS was used to label Mincle protein in RAW264.7 cell stimulated by TDB for 24 h. The results further confirmed that 3 μg/ml BITC effectively decreased the level of Mincle protein that stimulated by TDB (Figures 8A,B; p < 0.001).