Rosmarinic acid alleviates fungal keratitis caused by Aspergillus fumigatus by inducing macrophage autophagy.

Fungal keratitis (FK) is an infectious keratopathy can cause serious damage to vision. Its severity is related to the virulence of fungus and response of inflammatory. Rosmarinic acid (RA) extracted from Rosmarinus officinalis exhibits antioxidant, anti-inflammatory and anti-viral properties. The aim of this study was to investigate the effect of RA on macrophage autophagy and its therapeutic effect on FK. In this study, we demonstrated that RA reduced expression of proinflammatory cytokine, lessened the recruitment of inflammatory cells in FK. The relative contents of autophagy markers, such as LC3 and Beclin-1, were significantly up-regulated in RAW 264.7 cells and FK. In addition, RA restored mitochondrial membrane potential (MMP) of macrophage to normal level. RA not only reduced the production of intracellular reactive oxygen species (ROS) but also mitochondria ROS (mtROS) in macrophage. At the same time, RA induced macrophage to M2 phenotype and down-regulated the mRNA expression of IL-6, IL-1ß, TNF-α. All the above effects could be offset by the autophagy inhibitor 3-Methyladenine (3-MA). Besides, RA promote phagocytosis of RAW 264.7 cells and inhibits spore germination, biofilm formation and conidial adherence, suggesting a potential therapeutic role for RA in FK.

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.

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.

Honokiol reduces fungal burden and ameliorate inflammation lesions of Aspergillus fumigatus keratitis via Dectin-2 down-regulation.

PURPOSE: To screen and identify the mechanism of honokiol on anti-fungi and anti-inflammation in fungal keratitis (FK) through bioinformatic analysis and biological experiments. METHODS: Transcriptome profile demonstrated differential expression genes (DEGs) of Aspergillus fumigatus keratitis between PBS-treated and honokiol-treated groups via bioinformatics analyses. Inflammatory substances were quantified by qRT-PCR, Western blot and ELISA, and macrophage polarization was examined by flow cytometry. Periodic acid Schiff staining and morphological interference assay were used to detect hyphal distribution in vivo and fungal germination in vitro, respectively. Electron microscopy was to illustrate hyphal microstructure. RESULTS: Illumina sequencing demonstrated that compared with the honokiol group, 1175 up-regulated and 383 down-regulated genes were induced in C57BL/6 mice Aspergillus fumigatus keratitis with PBS treatment. Through GO analysis, some differential expression proteins (DEPs) played major roles in biological processes, especially fungal defense and immune activation. KEGG analysis provided fungus-related signaling pathways. PPI analysis demonstrated that DEPs from multiple pathways form a close-knit network, providing a broader context for FK treatment. In biological experiments, Dectin-2, NLRP3 and IL-1ß were upregulated by Aspergillus fumigatus to evaluate immune response. Honokiol could reverse the trend, comparable to Dectin-2 siRNA interference. Meanwhile, honokiol could also play an anti-inflammatory role via promoting M2 phenotype polarization. Moreover, honokiol reduced hyphal distribution in the stroma, delayed germination, and destroyed the hyphal cell membrane in-vitro. CONCLUSIONS: Honokiol possesses anti-fungal and anti-inflammatory effects in Aspergillus fumigatus keratitis and may develop a potential and safe therapeutic modality for FK.

The therapeutic role and mechanism of 4-Methoxycinnamic acid in fungal keratitis.

Fungal keratitis is an infectious vision-threatening disease that has a poor prognosis, and the clinical therapeutic drugs have multiple limitations, such as epithelial toxicity and low bioavailability. Therefore, new antifungal treatment strategies must be developed. 4-Methoxycinnamic acid (MCA) is a widely occurring natural phenolic acid that has been proven to have multiple effects, such as antibacterial, antifungal, anti-inflammatory, neuroprotective, and inhibiting cancer. In this research, we explored the effects and underlying mechanisms of MCA on A. fumigatus keratitis and the antifungal effects of the combination of MCA and natamycin (NATA) on A. fumigatus. We found that MCA exerts antifungal effects by inhibiting the synthesis of the fungal cell wall, changing the permeability of fungal cell membranes. Moreover, the MCA-NATA combination exhibited synergy for A. fumigatus. In addition, MCA exerted an anti-inflammatory effect by downregulating the inflammatory factors (IL-1ß, TNF-α, IL-6, and iNOS) in C57BL/6 mice and RAW264.7 cells. The anti-inflammatory mechanism of MCA was associated with the Mincle signal pathway. In summary, MCA acts as a potential therapeutic drug for fungal keratitis and a potential antifungal sensitizer for natamycin. MCA inhibits fungal cell wall synthesis, destroys the permeability of fungal cell membranes, and mediates the anti-inflammatory, immune response of the host.

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.

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.

Mechanism of antifungal activity and therapeutic action of ß-ionone on Aspergillus fumigatus keratitis via suppressing LOX1 and JNK/p38 MAPK activation.

PURPOSE: To investigate the anti-inflammatory and antifungal role of ß-ionone (BI) in fungal keratitis (FK). METHODS: In vitro antifungal activity of BI against Aspergillus fumigatus (A. fumigatus) was evaluated by using minimum inhibitory concentration (MIC), crystal violet staining, biofilm biomass measurement, propidium iodide uptake test, and adherence assay. And RT-PCR was carried out to measure the levels of RodA, RodB, Rho, FKs, CshA-D, RlmA, Cyp51A-B and Cdr1B. Network pharmacology analysis was applied to predict the relationship between BI and FK. Cell Count Kit-8 (CCK8) assay was utilized to detect the cytotoxicity of BI to RAW264.7 and immortalized human corneal epithelial cells (HCECs). The underlying mechanism of BI at regulating the level of inflammatory factors in FK was assessed by RT-PCR, ELISA and Western blot in vitro and in vivo. The therapeutic effect of BI has investigated in A. fumigatus keratitis by employing the clinical score, pathological examination, plate count, immunofluorescence and myeloperoxidase (MPO) assay. We also used the slit-lamp microscopy, clinical scores, and HE staining to assess the effect of natamycin compared with BI treatment in vivo. RESULTS: BI suppressed the growth of A. fumigatus and had a significant effect on A. fumigatus biofilms and membrane permeability. RT-PCR demonstrated that exposure of A. fumigatus to BI inhibited the expression of genes that function in hydrophobin (RodA, RodB), cell wall integrity (Rho, FKs, CshA-D, RlmA), azole susceptibility (Cyp51A-B, Cdr1B). Network pharmacology showed that the effects of BI in FK implicate with C-type lectin receptor signaling pathway. In vivo, after A. fumigatus infection, BI treatment markedly reduced the severity of FK by decreasing clinical score, neutrophil recruitment, and fungal load. And BI treatment also obviously reduced the expression of inflammatory cytokines, Lectin-like oxidized LDL receptor (LOX-1), phosphorylation of p38MAPK and p-JNK versus the DMSO-treated group. BI and natamycin both significantly increased corneal transparency and decreased inflammatory cell recruitment in the FK in the mice model. CONCLUSION: These results indicated that BI had fungicidal activities against A. fumigatus. It also ameliorated FK in mice by reducing inflammation, which was regulated by LOX-1, p-p38MAPK and p-JNK.

Mammalian Ste20-like kinase 4 inhibits the inflammatory response in Aspergillus fumigatus keratitis.

Mammalian Ste20-like kinase 4 (MST4), a new member of the germinal-center kinase STE20 family, was recently demonstrated to be a negative regulator of inflammation. However, whether MST4 participates in the inflammatory response to fungal infection remains unknown. Our study investigated the role and molecular mechanisms of MST4 in mice cornea and corneal epithelial cells exposed to Aspergillus fumigatus (A. fumigatus). Protein level of MST4 was detected in mice corneas and human corneal epithelial cells (HCECs) by Western blot analysis. The MST4 protein level was significantly elevated in mice corneas infected with A. fumigatus and HCECs exposed to A. fumigatus. MST4 expression was also detected in mice corneas by immunofluorescence staining. Furthermore, we found recombinant MST4 inhibited proinflammatory cytokines expressions induced by A. fumigatus at both the mRNA and protein levels in mice corneas and HCECs. To further investigate the mechanism of MST4's anti-inflammatory effect in A. fumigatus keratitis, we verified recombinant MST4 can inhibit curdlan-mediated proinflammatory cytokines production in HCECs. Surprisingly, recombinant MST4 protein downregulated A. fumigatus-induced Dectin-1 expression in both mRNA and protein levels in mice corneas. Recombinant MST4 can inhibit the mRNA expression level of Dectin-1 which was induced by curdlan in HCECs. MST4 can also inhibit the expression of Dectin-1 in mRNA levels increased by Dectin-1 overexpression plasmid in HCECs. Moreover, A. fumigatus or curdlan significantly induced the phosphorylation of Syk, which was consequently suppressed by recombinant MST4. Finally, recombinant MST4 promotes HCECs proliferation, which contribute to cornea wound healing. Taken together, our results provide evidences that MST4 inhibits inflammatory signaling response in A. fumigatus keratitis by downregulating Dectin-1/p-Syk pathway and simultaneously promotes HCECs proliferation.

The anti-inflammatory regulation of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

AIM: To analyze the impact of calcitonin gene-related peptide (CGRP) in mouse keratitis after Aspergillus fumigatus (A. fumigatus) infection. METHODS: C57BL/6 mice were treated subconjunctivally with different concentrations of exogenous CGRP, and BALB/c mice were treated with CGRP8-37 (a CGRP antagonist) before corneas were infected with A. fumigatus. The cornea was assessed under the slit-lamp and the clinical score was recorded. The mRNA levels of IL-1ß, TNF-α, IL-6, and MIP-2 were detected by quantitative real-time polymerase chain reaction (PCR), while the protein level of IL-1ß was determined by Western blotting. In vitro, RAW264.7 cells were used to investigate NLRP3 and IL-1ß expression induced by A. fumigatus after the pretreatment of exogenous CGRP or CGRP8-37. Cytokines expression in RAW264.7 cells was evaluated by real-time PCR and Western blotting. RESULTS: Using exogenous CGRP resulted in down-regulated synthesis of IL-1ß and MIP-2 stimulated by A. fumigatus in C57BL/6 mice keratitis, and the synthesis of IL-1ß, MIP-2 and IL-6 was up-regulated in BALB/c mice corneas after the pretreatment with CGRP8-37. Pretreatment with exogenous CGRP and CGRP8-37 did not influence TNF-α mRNA levels either in BALB/c or C57BL/6 mice keratitis. The levels of NLRP3 and IL-1ß were both reduced in A. fumigatus stimulated-macrophages after treatment with exogenous CGRP. And CGRP8-37 pretreatment would increase NLRP3 and IL-1ß levels. CONCLUSION: CGRP may alleviate the inflammatory reaction in mice keratitis after infection with A. fumigatus. The anti-inflammatory effect may be related to the inhibition of NLRP3 expression by CGRP.

FCN-A mediates the inflammatory response and the macrophage polarization in Aspergillus fumigatus keratitis of mice by activating the MAPK signaling pathway.

Fungal keratitis (FK) is a severe corneal disease that may cause vision loss. Previous studies indicate that the innate immune response produces the most effective anti-Aspergillus immune resistance. Ficolin-A (FCN-A), a soluble pattern-recognition receptor (PRR) family plays an important role in the innate immunity. In this study, we aimed to study the role of FCN-A in the A. fumigatus infected cornea. Here for the first time, we reported that the expression of FCN-A increases after A. fumigatus infection in the cornea of mice. Then, our results showed that the down-regulation of FCN-A reduced the inflammatory response of the cornea infected mice and decreased the expression of the TNF-a, p-p38, p-JNK. We also found that FCN-A can affect the recruitment of macrophages in the cornea of mice with A. fumigatus keratitis. In the mouse model of A. fumigatus keratitis and the A. fumigatus stimulation of RAW 264.7 cells, knocking down of FCN-A expression promoted the macrophage polarization toward M2. Furthermore, we observed that both the p38 and JNK inhibitors pretreatment decreased the proportion of M1/M2 in RAW 264.7 cells. Taken together, our data provide evidence that FCN-A participated in the inflammatory response of A. fumigatus keratitis in mice. Moreover, FCN-A mediates the inflammatory response and the polarization of the macrophages by activating the MAPK signaling pathway in A. fumigatus keratitis.

Expression and role of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

AIM: To investigate the expression and role of calcitonin gene-related peptide (CGRP) in the mouse models induced by Aspergillus fumigatus (A. fumigatus). METHODS: C57BL/6 mice were randomized into a control group and A. fumigatus keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction (PCR) and immunohistofluorescence analysis were applied to detect CGRP expression in cornea of both groups. In vitro, tests were conducted with C57BL/6 mice macrophages to investigate CGRP expression after interaction with A. fumigatus. Cytokines expression induced by exogenous CGRP and the antagonist CGRP8-37 in A. fumigatus-exposed macrophages was evaluated by real-time PCR and ELISA. RESULTS: The cornea expression of CGRP was significantly elevated in C57BL/6 mice corneas and macrophages after A. fumigatus infection. After treatment with exogenous CGRP, the levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were reduced, and IL-10 level was increased in the A. fumigatus stimulated-macrophages. However, IL-1ß, TNF-α and IL-6 levels were upregulated after pretreatment of CGRP8-37. But the mRNA levels of MIP-2, TGF-ß and IL-10 were not changed. CONCLUSION: This study provides evidence that A. fumigatus increased CGRP expression. CGRP may play a protective role against inflammation in A. fumigatus keratitis.

Pannexin 1 Channels Contribute to IL-1ß Expression via NLRP3/Caspase-1 Inflammasome in Aspergillus Fumigatus Keratitis.

Purpose: Pannexin 1 channels are deemed to play important roles in inflammation. However, there is limited information regarding their roles in fungal infection diseases, especially fungal keratitis. This study aimed to investigate the role of pannexin 1 channels in Aspergillus fumigatus (A. fumigatus) keratitis. Materials and Methods: Mouse models or immortalized human corneal epithelial cells (HCECs) were infected with or without A. fumigatus for given time. The expression of pannexin 1 channels was tested by qPCR, western blot and immunofluorescence staining. Mice of A. fumigatus keratitis were pretreated with carbenoxolone (CBX) or 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) to block or activate the opening of pannexin 1 channels respectively. The clinical score was recorded. Cornea tissues were examined for the downstream signals of pannexin 1 channels, including NLRP3, Caspase-1 and IL-1ß, and myeloperoxidase (MPO) by PCR and ELISA. Data were analyzed with commercial data analysis software and a P < 0.05 was considered to be statistically significant. Results: Upon A. fumigatus infection, pannexin 1 expression increased at both the mRNA and the protein levels in mice corneas (P< 0.05, n = 3). Immunofluorescence indicated that pannexin 1 channels were mainly located in the corneal epithelial layer, and they were upregulated after A. fumigatus infection. In vitro, the same tendency was found at the mRNA and the protein levels in HCECs (P< 0.05, n = 8). In mouse model, blockage of pannexin 1 channels by CBX caused more severely keratitis. The downstream signals of pannexin 1 channels (NLRP3/Caspase-1/IL-1ß) and MPO were down-regulated. Whereas activation the opening of pannexin 1 channels by BzATP reduced corneal infection with increased expression of Caspase-1 and IL-1ß. Conclusions: Pannexin 1 channels play important roles in the regulation of progression and leucocytes aggregation during corneal A. fumigatus infection via the NLRP3/Caspase-1/IL-ß pathway.

Organosilane and Polyethylene Glycol Functionalized Magnetic Mesoporous Silica Nanoparticles as Carriers for CpG Immunotherapy In Vitro and In Vivo.

Cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) have significant clinical potential as immunotherapeutics. However, limitations exist due to their transient biological stability in vivo, lack of specificity for target cells, and poor cellular uptake. To address these issues, we prepared amine magnetic mesoporous silica nanoparticles (M-MSN-A) then further modified with polyethylene glycol (PEG) for use as CpG delivery vectors. The PEG modified M-MSN-A (M-MSN-P) had notable CpG ODN loading capacity, negligible cytotoxicity, and were easily internalized into cells where they released the loaded CpG into the cytoplasm. As a result, such complexes were effective in activating macrophages and inhibiting tumor cells when combined with chemotherapeutics in vitro. Furthermore, these complexes had excellent immuno-stimulating activity in vivo, compared to the free CpG therapeutics. We report here a highly effective MSNs-based delivery system with great potential as a therapeutic CpG formulation in cancer immunotherapy.