Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

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.

Aspergillus fumigatus-Derived Extracellular Vesicles Mitigate Fungal Keratitis by Modulating the Immune Cell Function.

Fungal keratitis (FK) is a refractory global disease characterized by a high incidence of blindness and a lack of effective therapeutic options, and Aspergillus fumigatus (A. fumigatus, AF) is one of the most common causative fungi. This study aimed to investigate the role of extracellular vesicles (EVs) from A. fumigatus in the immune cell function and their protective role in A. fumigatus keratitis in order to explore their therapeutic potential. First, we isolated and characterized the EVs (AF-derived EVs). In vitro, we stimulated RAW264.7 cells and polymorphonuclear cells with AF-derived EVs. The expression levels of inflammatory factors increased in both immune cells along with an M1 polarization variation of RAW264.7 cells. After being incubated with AF-derived EVs, both immune cells exhibited an increased conidia-phagocytic index and a decreased conidia survival rate. In vivo, we injected EVs subconjunctivally on mice resulting in a heightened production of secretory immunoglobulin A (sIgA) in tear fluid. By the injection of EVs on mice in advance, a significant reduction in severity of A. fumigatus FK was witnessed by lower clinical scores, inflammatory appearances, and mitigated fungal load. Collectively, these results positioned AF-derived EVs as a promising and innovative immune therapy for combating FK, while also providing a platform for further investigation into developing an optimal formulation for modulating inflammation in the context of FK.

Localized calcium oxalate crystals in primary cutaneous aspergillosis.

Select Aspergillus species can produce oxalate as a fermentation byproduct, which may react with calcium ions to produce insoluble calcium oxalate crystals in tissues. These crystals are frequently associated with pulmonary Aspergillus infections, yet are rarely described in primary cutaneous aspergillosis. Herein, we report the presence of calcium oxalate crystals detected on cutaneous specimens from primary cutaneous Aspergillus niger and Aspergillus fumigatus infections in an immunocompromised, premature infant. No metabolic sources of oxalosis were found.

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.

Pseudolaric Acid B Ameliorates Fungal Keratitis Progression by Suppressing Inflammation and Reducing Fungal Load.

This study aimed to determine the mechanisms of antifungal and anti-inflammation effects of pseudolaric acid B (PAB) on Aspergillus fumigatus (A. fumigatus) keratitis. In vitro MIC assay and crystal violet staining were conducted to evaluate the efficacy of PAB against A. fumigatus. PAB inhibited A. fumigatus growth and inhibited the formation of fungal biofilms in a dose-dependent manner. Molecular docking analysis revealed that PAB possesses strong binding properties with Rho1 of A. fumigatus, which is devoted to encoding (1,3)-ß-d-glucan of A. fumigatus. RT-PCR results also showed that Rho1 was inhibited by PAB. In vivo, PAB treatment reduced clinical scores, fungal load, and macrophage infiltration, which were increased by A. fumigatus in mice corneas. In addition, PAB treatment suppressed the expression of Mincle, p-Syk, and cytokines (TNF-α, MIP2, iNOS, and CCL2) in infected corneas and in RAW264.7 cells, which were tested by RT-PCR, Western blot, and enzyme-linked Immunosorbent Assay. Notably, trehalose-6,6-dibehenate, an agonist of Mincle, pretreatment reversed the regulatory function of PAB in RAW 264.7 cells. Moreover, flow cytometry showed that PAB upregulated the ratio of M2/M1 macrophages in A. fumigatus-infected corneas and RAW264.7 cells. In conclusion, PAB produced antifungal activities against A. fumigatus and decreased the inflammatory response in mouse A. fumigatus keratitis.

Glabridin Inhibits Aspergillus fumigatus Growth and Alleviate Inflammation Mediated by Dectin-2 and NLRP3 Inflammasome.

PURPOSE: The research was used to uncover the mechanism of glabridin in Aspergillus fumigatus keratitis in anti-fungus and anti-inflammation. METHODS: In vitro, RAW 264.7 cells were infected with A. fumigatus with incubation of glabridin in different concentrations. Real-time quantitative polymerase chain reaction (RT­qPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA) were used to assess the inflammatory severe and alternation with the intervention of Dectin-2 siRNA and glabridin. In vivo, A. fumigatus keratitis mouse models were established by spore intra-stromal injection and treated with glabridin or PBS. And disease scores, inflammatory mediators, and periodic acid-schiff (PAS) staining were exhibited to demonstrate the therapeutic efficiency of glabridin in vivo. Morphological interference assay monitored fungal germination. Scanning and transmission electron microscopy were used to observe the growth of fungi. RESULTS: In RAW 264.7 cells and mouse keratitis models, noncytotoxic 16 µg/mL glabridin showed significant inhibition in the expression of Dectin-2, NLRP3, Caspase-1, IL-1ß, and TNF-α after A. fumigatus infection, almost similar to the intervention of Dectin-2 siRNA. PAS staining illustrated the reduced hyphal distribution in cornea stroma with glabridin treatment. Glabridin remarkably inhibited A. fumigatus growth through delaying germination and disrupting the integrity of the hyphae membrane. CONCLUSION: Glabridin plays an anti-inflammatory role in A. fumigatus challenge via suppression of the Dectin-2 and NLRP3 inflammasome, and plays an anti-fungal role through delaying germination and changing the hyphal integrity.KEY MESSAGESGlabridin plays an anti-inflammatory role in A. fumigatus infection of RAW264.7 cells in a concentration-dependent manner and through Dectin-2 mediation.Glabridin decreases fungal distribution and inflammation in mouse A. fumigatus keratitis.Glabridin inhibits A. fumigatus growth by delaying germination and disrupting cellular structure in vitro.

Pentoxifylline treats Aspergillus fumigatus keratitis by reducing fungal burden and suppressing corneal inflammation.

Fungal keratitis (FK) is a blinding ocular disease, which mainly results from fungal damage and excessive inflammation. Pentoxifylline, a kind of methylxanthine, has been discovered to have anti-inflammatory properties in various infectious diseases, hinting a potential therapeutic effect on treating corneal fungal infection. Whereas, the therapeutic impact of pentoxifylline on fungal keratitis is still uncertain. This study investigated the antifungal capability against Aspergillus fumigatus and the anti-inflammatory role of pentoxifylline by activating nuclear factor, erythroid 2 like 2 (Nrf2)/heme oxygenase1 (HO1) pathway in the process of FK. In our research, we demonstrated that pentoxifylline could effectively inhibit fungal growth and inflammatory reaction. Pentoxifylline reduced the production of pro-inflammatory factors by stimulating the Nrf2/HO1 pathway. Although there was no statistical difference between the curative efficacy of pentoxifylline and natamycin application to FK, pentoxifylline could promote corneal epithelial repair and was less toxicity to the ocular surface than natamycin. In conclusion, pentoxifylline performs antifungal and anti-inflammatory effects by lessening the fungus burden and activating the Nrf2/HO1 pathway, hinting that it has the potential to be a new therapeutic medication for Aspergillus fumigatus keratitis.

Dimethyl fumarate ameliorates fungal keratitis by limiting fungal growth and inhibiting pyroptosis.

PURPOSE: We aimed to investigate the therapeutic role of dimethyl fumarate (DMF) in fungal keratitis. METHODS: Human corneal epithelial cells (HCECs) and mouse models of fungal keratitis were used in this study. The antifungal effect of DMF on Aspergillus fumigatus (A. fumigatus) was confirmed by examining the minimum inhibitory concentration (MIC), biofilm formation, conidial adherence and corneal fungal loads. Slit-lamp photography, haematoxylin and eosin staining and immunostaining were used to assess the severity of corneal impairment. RT-PCR, western blot, ELISA, immunohistochemistry and immunostaining were performed to examine the effects of DMF on the expression of the inflammatory mediators during fungal infection. RESULTS: In vitro, DMF limited A. fumigatus growth, biofilm formation, and conidial adherence and reduced the mRNA levels of AldA, GlkA, GAPDH, HxkA, PgkA, Sdh2, GelA and ChsF in A. fumigatus. In vivo, DMF effectively decreased corneal fungal loads. DMF attenuated corneal inflammatory impairment by suppressing inflammatory cell accumulation and downregulating cytokine expression. DMF notably downregulated the high expression of NLRP3, cleaved GSDMD, cleaved caspase-1, mature IL-1ß and mature IL-18 induced by fungi. The production of Nrf2 and HO-1 could be further increased by DMF in infected HCECs. Nrf2 siRNA pretreatment counteracted DMF-mediated downregulation of the expression of the active forms of IL-18, IL-1ß, caspase-1 and GSDMD. CONCLUSION: DMF limits fungal growth by suppressing biofilm formation, conidial adherence and respiratory metabolism. It also exerts an anti-inflammatory effect on fungal keratitis by inhibiting pyroptosis, which could be regulated by Nrf2. Our results suggest that DMF plays a therapeutic role in fungal keratitis.

Thymol Ameliorates Aspergillus fumigatus Keratitis by Downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß Signal Expression and Reducing Necroptosis and Pyroptosis.

Fungal keratitis is a refractory kind of keratopathy. We attempted to investigate the anti-inflammatory role of thymol on Aspergillus fumigatus (A. fumigatus) keratitis. Wound healing and fluorescein staining of the cornea were applied to verify thymol's safety. Mice models of A. fumigatus keratitis underwent subconjunctival injection of thymol. The anti-inflammatory roles of thymol were verified by hematoxylin-eosin (HE) staining, slit lamp observation, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. In contrast with the DMSO group, more transparent corneas and less inflammatory cells infiltration were detected in mice treated with 50 µg/ml thymol. Thymol downregulated the synthesis of TLR4, MyD88, NF-kB, IL-1ß, NLRP3, caspase 1, caspase 8, GSDMD, RIPK3 and MLKL. In summary, we proved that thymol played a protective part in A. fumigatus keratitis by cutting down inflammatory cells aggregation, downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß signal expression and reducing necroptosis and pyroptosis.

Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response.

Purpose: The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1. Results: In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1ß, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control. Conclusions: GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.

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.

MCPIP1 alleviates inflammatory response through inducing autophagy in Aspergillus fumigatus keratitis.

Fungal keratitis (FK) is a serious corneal infection caused by pathogenic fungi. Monocyte chemoattractant protein-induced protein 1 (MCPIP1) plays an important role in restricting the inflammatory response in various immune disorders. However, the function of MCPIP1 in Aspergillus fumigatus (A. fumigatus) keratitis is unclear. In the present study, we found that A. fumigatus infection promotes the expression of MCPIP1 in human corneal epithelial cells (HCECs) and in mouse corneas. Overexpression of MCPIP1 decreased the production of inflammatory cytokines, including TNF-α, IL-6, and IL-1ß, while knockdown of MCPIP1 increased the expression of these cytokines. MCPIP1 enhanced autophagy flux by inhibiting the mTOR signaling in HCECs with A. fumigatus infection. Further study showed that inhibition of autophagy using chloroquine reverses the anti-inflammatory effect of MCPIP1 in HCECs infected with A. fumigatus. Moreover, MCPIP1 alleviated the severity of keratitis and inhibited the expression of inflammatory cytokines by activating autophagy in an FK mouse model. In conclusion, our study demonstrated that MCPIP1 alleviates the severity of A. fumigatus keratitis by inducing mTOR-mediated autophagy in HCECs and in a mouse model. Exogenous use of MCPIP1 protein may have potential applications in FK clinical therapy.

Oxidized chondroitin sulfate eye drops ameliorate the prognosis of fungal keratitis with anti-inflammatory and antifungal effects.

Fungal keratitis (FK) is a refractory ophthalmic disease that can result in vision impairment and even blindness due to the severe fungal invasiveness and excessive inflammatory response. Therefore, antifungal treatment combined with local immunosuppressive therapy is regarded as the most effective strategy to improve the clinical outcome of FK. Oxidized polysaccharides with aldehyde groups possess obvious inhibitory activity towards microorganisms. Herein, we use chondroitin sulfate (CS), a recognized anti-inflammatory biopolysaccharide, to prepare oxidized chondroitin sulfate (OCS) via sodium periodate (NaIO4) oxidation for the treatment of FK. The chemical structure of OCS was characterized by FTIR, 1H NMR, and XPS, revealing that the O-dihydroxy in the D-glucuronic acid unit of CS was selectively broken by NaIO4, forming active aldehyde groups. The introduction of aldehydes not only retains the anti-inflammatory activity but also confers OCS with antifungal property. In vitro antifungal experiments showed that OCS inhibits the growth, represses the biofilm formation and alters the membrane integrity of A. fumigatus. The toxicity of OCS was evaluated by cytotoxicity tests (CCK-8) and the Draize eye test in vitro and in vivo. qRT-PCR confirmed that OCS had similar anti-inflammatory activity as CS. In mice with A. fumigatus keratitis, OCS versus CS or PBS showed an excellent therapeutic effect, characterized by a lower corneal inflammation score, less fungal load, reduced neutrophil recruitment, and the downregulated expression of pro-inflammatory factors. Our findings demonstrate that OCS improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of fungi, reducing the recruitment of neutrophils and inhibiting the inflammatory response. It provides innovative ideas for the development and application of OCS in medicine and biomaterials fields.

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.

Increased susceptibility of irradiated mice to Aspergillus fumigatus infection via NLRP3/GSDMD pathway in pulmonary bronchial epithelia.

BACKGROUND: Aspergillus fumigatus infection is difficult to diagnose clinically and can develop into invasive pulmonary aspergillosis, which has a high fatality rate. The incidence of Aspergillus fumigatus infection has increased die to widespread application of radiotherapy technology. However, knowledge regarding A. fumigatus infection following radiation exposure is limited, and the underlying mechanism remains unclear. In this study, we established a mouse model to explore the effect of radiation on A. fumigatus infection and the associated mechanisms. METHODS: In this study, a mouse model of A. fumigatus infection after radiation was established by irradiating with 5 Gy on the chest and instilling 5 × 107/ml Aspergillus fumigatus conidia into trachea after 24 h to explore the effect and study its function and mechanism. Mice were compared among the following groups: normal controls (CON), radiation only (RA), infection only (Af), and radiation + infection (RA + Af). Staining analyses were used to detect infection and damage in lung tissues. Changes in protein and mRNA levels of pyroptosis-related molecules were assessed by western blot analysis and quantitative reverse transcription polymerase chain reaction, respectively. Protein concentrations in the serum and alveolar lavage fluid were also measured. An immunofluorescence colocalization analysis was performed to confirm that NLRP3 inflammasomes activated pyroptosis. RESULTS: Radiation destroyed the pulmonary epithelial barrier and significantly increased the pulmonary fungal burden of A. fumigatus. The active end of caspase-1 and gasdermin D (GSDMD) were highly expressed even after infection. Release of interleukin-18 (IL-18) and interleukin-1ß (IL-1ß) provided further evidence of pyroptosis. NLRP3 knockout inhibited pyroptosis, which effectively attenuated damage to the pulmonary epithelial barrier and reduced the burden of A. fumigatus. CONCLUSIONS: Our findings indicated that the activation of NLRP3 inflammasomes following radiation exposure increased susceptibility to A. fumigatus infection. Due to pyroptosis in lung epithelial cells, it resulted in the destruction of the lung epithelial barrier and further damage to lung tissue. Moreover, we found that NLRP3 knockout effectively inhibited the pyroptosis and reducing susceptibility to A. fumigatus infection and further lung damage. Overall, our results suggest that NLRP3/GSDMD pathway mediated-pyroptosis in the lungs may be a key event in this process and provide new insights into the underlying mechanism of infection. Video abstract.

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.

Eugenol protects against Aspergillus fumigatus keratitis by inhibiting inflammatory response and reducing fungal load.

Fungal keratitis is a corneal infection, which severely impairs vision. The fungal pathogen provokes host immune response, but the excessive inflammatory response causes significant collateral damage to the cornea. Eugenol, the main component of clove oil, has been found to have a broad range of pharmacological activities including anti-microbial, antioxidation and anti-inflammation. However, the role of eugenol in Aspergillus fumigatus (A. fumigatus) keratitis is unknown. In this study, we demonstrated that eugenol reduced mice keratitis severity, inflammatory cells infiltration, pro-inflammatory cytokine expression, and the fungal load. Eugenol also decreased the expressions of pro-inflammatory cytokines in human corneal epithelial cells (HCECs). We confirmed that the anti-inflammatory effects of eugenol were related to activating nuclear factor erythroid 2-related factor 2/Heme Oxygenase-1 (Nrf2/HO-1) signaling pathway. Moreover, we demonstrated that eugenol could inhibit the A. fumigatus growth and adhesion to host cells, as well as damage the fungal biofilm. The antifungal mechanism seemed to be disrupting the integrity of the fungal membrane and reducing the biosynthesis of ergosterol. Taken together, our research suggested that eugenol exerted protective effects on mouse A. fumigatus keratitis, due to its anti-inflammatory and antifungal activity.

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.

BTK Inhibitors Impair Platelet-Mediated Antifungal Activity.

In recent years, the introduction of new drugs targeting Bruton's tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.