The Role of LC3-Associated Phagocytosis Inhibits the Inflammatory Response in Aspergillus fumigatus Keratitis.

Purpose: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis. Methods: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1ß, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence. Results: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1ß and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody. Conclusions: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

Involvement of cGAS/STING Signaling in the Pathogenesis of Candida albicans Keratitis: Insights From Genetic and Pharmacological Approaches.

Purpose: Fungal keratitis (FK) is an invasive corneal infection associated with significant risk to vision. Although the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway has been recognized for its role in defending against viral infections, its involvement in FK still remains largely unclear. This study sought to elucidate the contribution of the cGAS/STING signaling pathway to the pathogenesis of FK. Methods: The expression of cGAS/STING signaling components was assessed in a murine model of Candida albicans keratitis through RNA sequencing, western blot analysis, immunofluorescence staining, and real-time PCR. Both genetic (utilizing Sting1gt/gt mice) and pharmacological (using C176) interventions were employed to inhibit STING activity, allowing for the evaluation of resultant pathogenic alterations in FK using slit-lamp examination, clinical scoring, hematoxylin and eosin (H&E) staining, fungal culture, and RNA sequencing. Subconjunctival administration of the NOD-like receptor protein 3 (NLRP3) inflammasome inhibitor MCC950 was performed to evaluate FK manifestations following STING activity blockade. Furthermore, the impact of the STING agonist diABZI on FK progression was investigated. Results: Compared to uninfected corneas, those infected with C. albicans exhibited increased expression of cGAS/STING signaling components, as well as its elevated activity. Inhibiting cGAS/STING signaling exacerbated the advancement of FK, as evidenced by elevated clinical scores, augmented fungal load, and heightened inflammatory response, including NLRP3 inflammasome activation and pyroptosis. Pharmacological inhibition of the NLRP3 inflammasome effectively mitigated the exacerbated FK by suppressing STING activity. Conversely, pre-activation of STING exacerbated FK progression compared to the PBS control, characterized by increased fungal burden and reinforced inflammatory infiltration. Conclusions: This study demonstrates the essential role of the cGAS/STING signaling pathway in FK pathogenesis and highlights the necessity of its proper activation for the host against FK.

Ebselen improves fungal keratitis through exerting anti-inflammation, anti-oxidative stress, and antifungal effects.

Fungal keratitis is a severely vision-threatening corneal infection, where the prognosis depends on both fungal virulence and host immune defense. Inappropriate host responses can induce substantial inflammatory damage to the cornea. Therefore, in the treatment of fungal keratitis, it is important to concurrently regulate the immune response while efforts are made to eliminate the pathogen. Ebselen is a widely studied organo-selenium compound and has been demonstrated to have antifungal, antibacterial, anti-inflammatory, and oxidative stress-regulatory properties. The effectiveness of ebselen for the treatment of fungal keratitis remains unknown. In this study, ebselen was demonstrated to produce a marked inhibitory effect on Aspergillus fumigatus (A. fumigatus), including spore germination inhibition, mycelial growth reduction, and fungal biofilm disruption. The antifungal activity of ebselen was related to the cell membrane damage caused by thioredoxin (Trx) system inhibition-mediated oxidative stress. On the contrary, ebselen enhanced the antioxidation of Trx system in mammalian cells. Further, ebselen was proven to suppress the expressions of inflammatory mediators (IL-1ß, IL-6, TNF-α, COX-2, iNOS, and CCL2) and reduce the production of oxidative stress-associated indicators (ROS, NO, and MDA) in fungi-stimulated RAW264.7 cells. In addition, ebselen regulated PI3K/Akt/Nrf2 and p38 MAPK signaling pathways, which contributed to the improvement of inflammation and oxidative stress. Finally, we verified the therapeutic effect of ebselen on mouse fungal keratitis. Ebselen improved the prognosis and reduced the fungal burden in mouse corneas. Expressions of inflammatory mediators, as well as the infiltration of macrophages and neutrophils in the cornea were also obviously decreased by ebselen. In summary, ebselen exerted therapeutic effects by reducing fungal load and protecting host tissues in fungal keratitis, making it a promising treatment for fungal infections.

Mesoporous zinc oxide-based drug delivery system offers an antifungal and immunoregulatory strategy for treating keratitis.

Fungal keratitis is a refractory eye disease that is prone to causing blindness. Fungal virulence and inflammatory responses are two major factors that accelerate the course of fungal keratitis. However, the current antifungal drugs used for treatment usually possess transient residence time on the ocular surface and low bioavailability deficiencies, which limit their therapeutic efficacy. In this work, natamycin (NATA)-loaded mesoporous zinc oxide (Meso-ZnO) was synthesized for treating Aspergillus fumigatus keratitis with excellent drug-loading and sustained drug release capacities. In addition to being a carrier for drug delivery, Meso-ZnO could restrict fungal growth in a concentration-dependent manner, and the transcriptome analysis of fungal hyphae indicated that it inhibited the mycotoxin biosynthesis, oxidoreductase activity and fungal cell wall formation. Meso-ZnO also promoted cell migration and exhibited anti-inflammatory role during fungal infection by promoting the activation of autophagy. In mouse models of fungal keratitis, Meso-ZnO/NATA greatly reduced corneal fungal survival, alleviated tissue inflammatory damage, and reduced neutrophils accumulation and cytokines expression. This study suggests that Meso-ZnO/NATA can be a novel and effective treatment strategy for fungal keratitis.

Oxymatrine mitigates Aspergillus fumigatus keratitis by suppressing fungal activity and restricting pyroptosis.

Fungal keratitis (FK) is a refractory keratitis caused by excessive inflammation and fungal damage. Excessive inflammation can lead to tissue damage and corneal opacity, resulting in a poor prognosis for FK. Oxymatrine (OMT) is a natural alkaloid, which has rich pharmacological effects, such as antioxidant and anti-inflammation. However, its antifungal activity and the mechanism of action in FK have not been elucidated. This study confirmed that OMT suppressed Aspergillus fumigatus growth, biofilm formation, the integrity of fungal cell and conidial adherence. OMT not only effectively reduced corneal fungal load but also inflammation responses. OMT lessened the recruitment of neutrophils and macrophages in FK. In addition, OMT up-regulated the expression of Nrf2 and down-regulated the expression of IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD. Pre-treatment with Nrf2 inhibitor up-regulated the expression of IL-1ß, IL-18, caspase-1, NLRP3 and GSDMD supressed by OMT. In conclusion, OMT has efficient anti-inflammatory and antifungal effects by suppressing fungal activity and restricting pyroptosis via Nrf2 pathway. OMT is considered as a potential option for the treatment of FK.

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

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-1ß. 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-1ß expression; and provided a potential treatment for fungal keratitis. Translational Relevance: This study provides a potential treatment for fungal keratitis in the clinic.

The Biochemical Characteristics in Experimental Keratomycosis.

PURPOSE: To investigate the biochemical characteristics in experimental keratomycosis. METHODS: Experimental mice were injected with Fusarium solanum solution Controls mice received liposomes containing phosphate-buffered saline (PBS-LIP). Raman spectroscopy was used to analyze the biochemical characteristics. The infiltration of inflammatory cells was analyzed by histopathology. Cytokine mRNA levels were detected by real-time polymerase chain reaction. RESULTS: Raman Spectroscopy: In the experimental group, collagen, lipids, amide I and III were decreased, amide II, hyper proline amino acids, and arginine were increased, and proline and phenylalanine were significantly increased on day 3. Histopathology: more severe keratomycosis developed in the experimental group than in the control group. Statistically significant mRNA expression of Collagen4\MMP2\MMP9\TIMP1.MMP9 was negatively correlated with the secretion of Collagen4. CONCLUSIONS: Matrix metalloproteinases are involved in biochemical changes in keratomycosis.

Aquaporin 5-in Extracellular Vesicles of Human Vitreous as a Potential Marker for Fungal Endophthalmitis.

PURPOSE: Extracellular vesicles (EVs) are lipid-bilayered nanoparticles that play an important role in cellular cross-talk, and as received attention for their role as diseases biomarker. Aquaporin-5 (AQP5) is a small integral membrane protein that help in the migration of cells, proliferation, and invasion. However, the association of AQP5 with fungal diseases is still unknown. The aim of this study was to evaluate the expression of AQP5 in EVs (EV-AQP5) extracted from the vitreous of patients with Fungal Endophthalmitis (FE). METHODS: Vitreous fluid was collected from 20 patients clinically suspected as FE, 10 patients from non-infectious conditions, and 10 patients with bacterial endophthalmitis as controls. EVs were isolated from human vitreous and characterized by dynamic light scattering, and scanning electron microscopy. Human Aquaporin-5 levels were evaluated using a commercial ELISA Kit. The Receiver Operating Characteristic (ROC) curves and its significance were correlated with microbiology data. RESULTS: Isolated EVs size were approx.250-380 nm in diameter. The measured levels of EV-AQP5 resulted significantly higher in FE patients (mean=216±15pg/ml; 95% confidence interval (CI): 182-250) in comparison to controls (mean=130±12pg/ml; 95%CI: 111-166)(p = .001). However, AQP5 levels in EVs derived from culture-proven bacteria patients were insignificant compared to controls (mean=169±4 pg/ml; 95%CI: 161-177). ROC curve was used to define the optimal cut-off level of the test at 180 pg/ml with an AUC of 98% (95%CI: 95-100) (p = .03), with a sensitivity of 100% and specificity of 90%. Additionally, the AQP5 level in EVs derived from culture-negative vitreous was above the threshold value (200 ± 10 pg/ml (95%CI: 180-230) in comparison to the control group (p < .001) However, no significant association was found between age or visual acuity and the level of AQP5 in FE. CONCLUSION: Our results reveal that the vitreous EV-AQP5 levels can aid in differentiating FE from non-infectious retinal conditions, mainly when the cultures are negative.

Flavopiridol Protects against Fungal Keratitis due to Aspergillus fumigatus by Alleviating Inflammation through the Promotion of Autophagy.

Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in Aspergillus fumigatus keratitis and explore its effects on autophagy. In our study, the corneas of the fungal keratitis mouse model were treated with 5 µM flavopiridol. In vitro, RAW 264.7 cells were pretreated with 200 nM flavopiridol before fungal stimulation. A. fumigatus was incubated with flavopiridol, and the antifungal activity of flavopiridol was detected. Our results indicated that flavopiridol treatment notably reduced clinical scores as well as cytokines expression of infected corneas. In infected RAW 264.7 cells, flavopiridol treatment inhibited IL-1ß, IL-6, and TNF-α expression but promoted IL-10 expression. Transmission electron microscopy (TEM) images showed that more autolysosomes were present in infected corneas and RAW 264.7 cells after flavopiridol treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1, and Atg-7. 3-Methyladenine (3-MA, an inhibitor of autophagy) pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. In conclusion, flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.

METTL3 Attenuates Inflammation in Fusarium solani-Induced Keratitis via the PI3K/AKT Signaling Pathway.

Purpose: Our previous investigations revealed a significant role of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification in the development of corneal inflammation in Fusarium infection, but the exact mechanism is unknown. Therefore, this research aimed to explore how METTL3 affects the inflammatory process of fungal keratitis (FK) in mice. Methods: We established in vitro and in vivo models by inoculating mice and primary corneal stromal cells with F. solani. METTL3 expression was confirmed by real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. After that, siRNAMETTL3 and AAV-sh-METTL3 were transfected into cells and mice to explore the role of METTL3 in the PI3K/AKT signaling pathway and inflammation. PI3K, p-PI3K, AKT, and p-AKT expression was analyzed by western blotting. Viability of corneal stromal cells was measured using a Cell Counting Kit-8 (CCK-8). Additionally, we detected interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α) levels in corneal tissues and analyzed the role of METTL3 in inflammation in FK using slit-lamp biomicroscopy and hematoxylin and eosin staining. Results: Here, our results show that METTL3 increased in mouse FK, and the expression of p-PI3K and p-AKT decreased when METTL3 was downregulated. We also found that knockdown of METTL3 expression attenuated the inflammatory response and decreased TNF-α, IL-1ß, and IL-6 expression in corneal-infected mice. Furthermore, inhibition of the PI3K/AKT pathway attenuated the inflammatory response of FK, decreased the expression of the above inflammatory factors, and enhanced the viability of corneal stromal cells. Conclusions: Based on the study results, METTL3 downregulation attenuates Fusarium-induced corneal inflammation via the PI3K/AKT signaling pathway.

Galectin-3 Is a Crucial Immunological Disease Marker in Patients with Fungal Keratitis.

Fungal keratitis, one of the most common infectious eye diseases in China, often results in a poor prognosis due to a delayed diagnosis and the insufficiency of effective therapy. There is an urgent need to identify specific biomarkers for the disease. In this study, we screened out tear proteins in patients with fungal keratitis by microsphere-based immunoassay analysis. Levels of cytokine expression were determined in both human corneal epithelial cell models in vitro and the corneas of patients by western blot, quantitative polymerase chain reaction (qPCR), and immunofluorescence analysis. Neutrophil activation was examined by flow cytometry analysis. The relationship between the cytokine expression and neutrophils was evaluated by immunofluorescence costaining and correlation analysis. These results demonstrated that the galectin-3 expression level was increased in both cell model and patient samples at the early and late stages of fungal keratitis. The neutrophils were significantly activated during the disease course of fungal keratitis. Meanwhile, colocalization and a positive correlation between galectin-3 and neutrophils were observed, suggesting that galectin-3 may play a crucial role in the recruitment of neutrophils and immune regulation of fungal keratitis. In conclusion, galectin-3 could be a key disease marker implying a beneficial immune response in the pathogenesis of fungal keratitis, which might be a target of therapeutic strategy in the future.

Laccase-mediated functionalization of natamycin by gallic acids for the therapeutic effect on Aspergillus fumigatus keratitis.

To improve the therapeutic effect of natamycin on fungal keratitis (FK), the grafted derivatives of natamycin and gallic acid were obtained, and the effects of the grafted derivatives on Aspergillus fumigatus (A. fumigatus) keratitis were investigated. The structure of natamycin grafted with gallic acid was identified by FT-IR and UV-Vis, and the successful synthesis of Gallic-Natamycin (GA-NAT) was proved. CCK-8 and the Draize eye test showed that GA-NAT had less cytotoxicity. Then, through in vitro antibacterial experiments such as minimum inhibitory concentration (MIC), adhesion, biofilm formation, and calcium fluorescence staining and in vivo experiments such as clinical score and plate counting, the results showed that GA-NAT had similar antifungal activity to natamycin, but had a better therapeutic effect than natamycin. Myeloperoxidase assay and immunofluorescence staining also showed that GA-NAT significantly inhibited neutrophil recruitment and activity. Moreover, It was further found that GA-NAT could inhibit the mRNA and protein expressions of LOX-1, TNF-α, and IL-1ß. These results indicated that GA-NAT inhibited the fungal growth, reduced the neutrophil infiltration into cornea, and down-regulated the expression of inflammatory factors in lesions, which provides a new choice for FK treatment.

Inhibition of Galectin-3 Impairs Antifungal Immune Response in Fungal Keratitis.

Galectin-3 is one of the galectin family members which are master regulators of immune homeostasis, especially in infectious diseases. However, its mechanism of immune regulation in fungal keratitis has not been thoroughly studied. Our study is aimed at clarifying the role of galectin-3 in the fungal keratitis mouse model in vivo, thereby providing a new biomarker of antifungal therapy. In our study, aspergillus, the most common pathogenic fungi of fungal keratitis, was identified and isolated by corneal tissue fungus culture. Then, the RNA expression levels of galectin family members in corneas of the mouse model with aspergillus fumigatus keratitis were screened by transcriptome sequencing (RNA-seq). The expression of the galectin-3 was detected by quantitative real-time Polymerase Chain Reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence in the corneal tissue of the fungal keratitis mouse model. Recruitment of neutrophils and the co-immunofluorescence of galectin-3 and related markers in corneal tissue were determined by flow cytometry analysis and immunofluorescence staining. The regulatory role of galectin-3 for proinflammatory cytokines and neutrophils was validated by the knockout mouse model. Galectin-3 knockout deteriorated the condition for the inhibition of galectin-3 was benefecial for fungi to survive and thrive in corneal lesions. These results demonstrated that in the ocular fungal infection, galectin-3 is capable of regulating the pathogenesis of fungal keratitis by modulating neutrophil recruitment. The deterioration of fungal keratitis and increased fungal load in corneal lesions of galectin-3 knockout mice proved the regulatory role of galectin-3 in fungal keratitis. In conclusion, galectin-3 is going to be an essential target to modulate neutrophil recruitment and its related antifungal immune response in fungal keratitis.

MiR-223-3p Regulates Autophagy and Inflammation by Targeting ATG16L1 in Fusarium solani-Induced Keratitis.

Purpose: Increasing evidence suggested that microRNAs (miRs) are implicated in the regulation of the inflammatory response and autophagy in multiple diseases. The present study aimed to explore the effect of miR-223-3p on inflammation and autophagy in fungal keratitis (FK). Methods: An FK mouse model was established, and primary corneal stromal cells were isolated by inoculation with Fusarium solani. The expression of miR-223-3p was determined by quantitative RT-PCR. Subsequently, the target gene of miR-223-3p was identified by a dual-luciferase reporter assay. The levels of miR-223-3p were altered by transfecting miR agomir/antagomir to evaluate its effects. Slit-lamp biomicroscopy and hematoxylin and eosin staining were employed to detect corneal damage. The levels of autophagy were assessed by immunofluorescence, Western blotting, mRFP-GFP-LC3 fluorescence microscopy, and electron microscopy. In addition, inflammation was demonstrated by determining the proinflammatory mediators IL-1ß and TNF-ɑ. Results: Our data suggested that miR-223-3p was increased and that autophagic flux was impaired in mouse FK. Then, we confirmed that autophagy-related gene 16L1 (ATG16L1) was a potential target of miR-223-3p and that this miR negatively regulated the expression of ATG16L1. The inhibition of miR-223-3p attenuated inflammation in FK, reduced P62 expression, and increased the ratio of LC3-II/LC3-I, whereas the overexpression of miR-223-3p displayed the opposite results. Conclusions: Taken together, miR-223-3p might regulate autophagy via targeting ATG16L1 in experimental F. solani keratitis and is associated with the inflammatory response. MiR-223-3p might be a potential therapeutic target for FK.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response.

Purpose: To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. Methods: The noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro. Results: Baicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1ß, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1ß, IL-6, and TNF-α were significantly lower in the TSLP siRNA-treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1ß, IL-6, and TNF-α. Conclusions: Baicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis.

Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.