Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway.

PURPOSE: The purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. METHODS: In order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit-lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 were determined using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and IF techniques. RESULTS: Schaftoside at a concentration of 160 µM displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1ß, TNF-α, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. CONCLUSIONS: Schaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.

Chelerythrine ameliorates Aspergillus fumigatus keratitis through suppressing the LOX-1/p38 MAPK signaling pathway.

PURPOSE: Aspergillus fumigatus (A. fumigatus) keratitis is a type of infectious corneal disease that significantly impairs vision. The objective of this study is to evaluate the therapeutic potential of chelerythrine (CHE) on A. fumigatus keratitis. METHODS: The antifungal activity of CHE was assessed through various tests including the minimum inhibitory concentration test, scanning electron microscopy, transmission electron microscopy, propidium iodide uptake test and plate count. Neutrophil infiltration and activity were assessed using immunofluorescence staining and the myeloperoxidase test. RT-PCR, western blotting assay, and ELISA were performed to measure the expression levels of proinflammatory cytokines (IL-1ß and IL-6), NF-E2-related factor (Nrf2), heme oxygenase-1 (HO-1), and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), as well as to determine the ratio of phosphorylated-p38 (p-p38) mitogen-activated protein kinase (MAPK) to p38 MAPK. RESULTS: In vitro, CHE inhibited the growth of A. fumigatus conidia, reduced fungal hyphae survival, and prevented fungal biofilm formation. In vivo, CHE reduced the severity of A. fumigatus keratitis and exhibited an excellent anti-inflammatory effect by blocking neutrophil infiltration. Furthermore, CHE decreased the expression levels of proinflammatory cytokines and LOX-1 at both mRNA and protein levels, while also decreasing the p-p38 MAPK/p38 MAPK ratio. Additionally, CHE increased the expression levels of Nrf2 and HO-1. CONCLUSION: CHE provides protection against A. fumigatus keratitis through multiple mechanisms, including reducing fungal survival, inducing anti-inflammatory effects, enhancing Nrf2 and HO-1 expression, and suppressing the signaling pathway of LOX-1/p38 MAPK.

Resveratrol has neuroprotective effects and plays an anti-inflammatory role through Dectin-1/p38 pathway in Aspergillus fumigatus keratitis.

PURPOSE: To determine the antifungal, anti-inflammatory and neuroprotective effects of resveratrol (RES) in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Cytotoxicity assay and Draize eye assay were performed to assess the toxicity of RES. The antifungal effect of RES was assessed by minimal inhibitory concentration, scanning or transmission electron microscopy, propidium iodide uptake assay, and Calcofluor white staining. Phosphorylation of p38 MAPK, mRNA and protein levels of Dectin-1 and related inflammatory factors were measured by qRT-PCR, ELISA and Western blot in vitro and in vivo. Clinical score, HE staining, plate count, and myeloperoxidase test were used to observe the progress of fungal keratitis. IF staining, qRT-PCR, and the Von Frey test were selected to assess the neuroprotective effects of RES. RESULTS: RES suppressed A. fumigatus hyphae growth and altered hyphae morphology in vitro. RES decreased the expression of Dectin-1, IL-1ß and TNF-α, as well as p38 MAPK phosphorylation expression, and also decreased clinical scores, reduced inflammatory cell infiltration and neutrophil activity, and decreased fungal load. RES also protected corneal basal nerve fibers, down-regulated mechanosensitivity thresholds, and increased the mRNA levels of CGRP and TRPV-1.. CONCLUSION: These evidences revealed that RES could exert antifungal effects on A. fumigatus and ameliorate FK through suppressing the Dectin-1/p38 MAPK pathway to down-regulate IL-1ß, IL-6, etc. expression and play protective effect on corneal nerves.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Comparative effectiveness of nitro dihydrocapsaicin, new synthetic derivative capsaicinoid, and capsaicin in alleviating oxidative stress and inflammation on lipopolysaccharide-stimulated corneal epithelial cells.

Loss of tear homeostasis, characterized by hyperosmolarity of the ocular surface, induces cell damage through inflammation and oxidation. Transient receptor potential vanilloid 1 (TRPV1), a sensor for osmotic changes, plays a crucial role as a calcium ion channel in the pathogenesis of hypertonic-related eye diseases. Capsaicin (CAP), a potent phytochemical, alleviates inflammation during oxidative stress events by activating TRPV1. However, the pharmacological use of CAP for eye treatment is limited by its pungency. Nitro dihydrocapsaicin (NDHC) was synthesized with aromatic ring modification of CAP structure to overcome the pungent effect. We compared the molecular features of NDHC and CAP, along with their biological activities in human corneal epithelial (HCE) cells, focusing on antioxidant and anti-inflammatory activities. The results demonstrated that NDHC maintained cell viability, cell shape, and exhibited lower cytotoxicity compared to CAP-treated cells. Moreover, NDHC prevented oxidative stress and inflammation in HCE cells following lipopolysaccharide (LPS) administration. These findings underscore the beneficial effect of NDHC in alleviating ocular surface inflammation, suggesting that NDHC may serve as an alternative anti-inflammatory agent targeting TRPV1 for improving hyperosmotic stress-induced ocular surface damage.

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.

Cytokine analysis of aqueous humor in patients with cytomegalovirus corneal endotheliitis.

PURPOSE: To evaluate cytokine levels of aqueous humor in patients with cytomegalovirus (CMV) corneal endotheliitis and their relationships with CMV DNA load. METHODS: 44 aqueous humor samples were obtained from 26 patients with CMV corneal endotheliitis at various stages of treatment. 33 samples obtained from cataract patients during the same period were selected as a control group. Each sample was used to measure the concentration of the CMV DNA load using real-time quantitative polymerase chain reaction, and to examine the levels of IL-6, IL-8, IL-10, MCP-1, VCAM-1, VEGF, IP-10, G-CSF, ICAM-1 and IFN-γ using a cytometric bead array. RESULTS: All 10 cytokines were found to have statistically significant differences between the CMV endotheliitis and cataract groups. The Spearman correlation test showed that the concentration of CMV DNA load was significantly associated with the levels of IL-6 (P = 0.005, r = 0.417), IL-8 (P < 0.001, r = 0.514), IL-10 (P < 0.001, r = 0.700), MCP-1 (P = 0.001, r = 0.487), VEGF (P < 0.001, r = 0.690), IP-10 (P = 0.001, r = 0.469), G-CSF (P < 0.001, r = 0.554) and ICAM-1 (P < 0.001, r = 0.635), but not significantly associated with VCAM-1 (P = 0.056) and IFN-γ (P = 0.219). CONCLUSIONS: There was a combined innate and adaptive immune response in aqueous humor in patients with CMV endotheliitis. Levels of multiple cytokines were significantly correlated with viral particle. Cytokines are potential indicators to help diagnose CMV endotheliitis, evaluate disease activity and assess treatment response.

Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release.

INTRODUCTION: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds. METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1ß, ß3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury. RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining. CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Pseudomonas aeruginosa-induced nociceptor activation increases susceptibility to infection.

We report a rapid reduction in blink reflexes during in vivo ocular Pseudomonas aeruginosa infection, which is commonly attributed and indicative of functional neuronal damage. Sensory neurons derived in vitro from trigeminal ganglia (TG) were able to directly respond to P. aeruginosa but reacted significantly less to strains of P. aeruginosa that lacked virulence factors such as pili, flagella, or a type III secretion system. These observations led us to explore the impact of neurons on the host's susceptibility to P. aeruginosa keratitis. Mice were treated with Resiniferatoxin (RTX), a potent activator of Transient Receptor Potential Vanilloid 1 (TRPV1) channels, which significantly ablated corneal sensory neurons, exhibited delayed disease progression that was exemplified with decreased bacterial corneal burdens and altered neutrophil trafficking. Sensitization to disease was due to the increased frequencies of CGRP-induced ICAM-1+ neutrophils in the infected corneas and reduced neutrophil bactericidal activities. These data showed that sensory neurons regulate corneal neutrophil responses in a tissue-specific matter affecting disease progression during P. aeruginosa keratitis. Hence, therapeutic modalities that control nociception could beneficially impact anti-infective therapy.

Quercetin protects against Aspergillus fumigatus keratitis by reducing fungal load and inhibiting TLR-4 induced inflammatory response.

PURPOSE: To investigate the antifungal and anti-inflammatory effects of quercetin in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Draize eye test was performed in mice to evaluate the toxicity of quercetin, and the antifungal effects on A. fumigatus were assessed via scanning electron microscopy (SEM), propidium iodide uptake, and adherence assay. In fungal keratitis (FK) mouse models, immunostaining was performed for investigating toll-like receptor 4 (TLR-4) expression and macrophage infiltration. Real-time PCR, ELISA, and Western blot were used to evaluate the expression of pro-inflammatory factors IL-1ß, TNF-α, and IL-6 in infected RAW264.7 cells. Cells were also treated with TLR-4 siRNA or agonist CRX-527 to investigate mechanisms underlying the anti-inflammatory activity of quercetin. RESULTS: Quercetin at 32 µM was non-toxic to corneal epithelial and significantly inhibited A. fumigatus growth and adhesion, and also altered the structure and reduced the number of mycelia. Quercetin significantly reduced macrophage infiltration in the mouse cornea, and attenuated the expression of TLR-4 in the corneal epithelium and stroma of mice with keratitis caused by A. fumigatus. In RAW264.7 cells infected by A. fumigatus, quercetin downregulated TLR-4 along with pro-inflammatory factors IL-1ß, TNF-α, and IL-6. RAW cells with TLR-4 knockdown had reduced expression of factors after A. fumigatus infection, which was decreased even further with quercetin treatment. In contrast, cells with CRX-527 had elevated inflammatory factors compared to control, which was significantly attenuated in the presence of quercetin. CONCLUSION: Quercetin plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal load, disrupting hyphae structure, macrophage infiltration, and suppressing inflammation response in macrophages via TLR-4 mediated signaling pathway.

Thymoquinone attenuates inflammation in C. Albicans keratitis by activating Nrf2/HO-1 signaling pathway and reducing fungal load.

PURPOSE: This study aims to investigate the anti-inflammatory and antifungal properties of thymoquinone (TQ) and elucidate its mechanism of action in the context of C. albicans keratitis. METHODS: Various methods were employed to identify a safe and effective concentration of TQ with antifungal properties, including the determination of the minimum inhibitory concentration (MIC), the cell counting kit-8 (CCK-8) test, and the Draize experiment. The severity of fungal keratitis (FK) was assessed through clinical ratings and slit-lamp imaging. Fungus burden was determined using plate counting and periodic acid Schiff (PAS) staining. Neutrophil infiltration and activity were investigated through immunofluorescence staining (IFS), myeloperoxidase (MPO) analysis, and hematoxylin and eosin (HE) staining. To explore the anti-inflammatory effects of TQ and its mechanism of action, we employed RT-PCR, ELISA, and western blot techniques. RESULTS: TQ effectively controlled fungal growth at a concentration of 50 µg/mL while preserving the integrity of mouse corneas. Human corneal epithelial cells (HCECs) remained unaffected by TQ at concentrations ≤ 3.75 µg/mL. Treatment with TQ led to significant improvements in clinical scores, fungal burden, neutrophil infiltration, and the expression of inflammatory factors compared to the DMSO group. Moreover, TQ demonstrated the ability to reduce the levels of inflammatory factors in HCECs stimulated by C. albicans. Additionally, TQ enhanced the expressions of Nrf2 and HO-1 in mouse corneas. The downregulation of cytokines induced by TQ was reversed upon pretreatment with inhibitors of Nrf2 or HO-1. CONCLUSION: TQ exhibits a protective effect in the context of C. albicans keratitis through multiple mechanisms, including inhibition of C. albicans growth, reduction of neutrophil recruitment, activation of the Nrf2/HO-1 pathway, and limitation of the expression of pro-inflammatory factors.

Therapeutic effect of Atractylenolide I on Aspergillus fumigatus keratitis by affecting MyD88/ NF-κB pathway and IL-1ß, IL-10 expression.

PURPOSE: Atractylenolide I (AT-I) is a natural sesquiterpene with anti-inflammatory effects. The purpose of this study was to research the anti-inflammatory effect of AT-I on Aspergillus fumigatus(A. fumigatus) keratitis in mice. METHODS: Cytotoxicity test and cell scratch test were used to determine the therapeutic concentrations of corneal infections. In vivo and in vitro studies, mouse cornea and human corneal epithelial cells (HCECs) infected with A. fumigatus were treated with AT-I or dimethyl sulfoxide (DMSO). Then, to analyze the effect of AT-I on inflammatory response, namely neutrophil or macrophage recruitment and the expression of cytokines involving MyD88, NF-κB, interleukin 1ß (IL-1ß) and interleukin 10 (IL-10). To study the effects of the drug, the techniques used include slit-lamp photography, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (QRT-PCR), and western blot. At the same time, in order to explore the combined effect of the drug and natamycin, slit-lamp photographs and clinical scores were used to visually display the disease process. RESULTS: No cytotoxicity was observed under the action of AT-I at a concentration of 800 µM. In mouse models, AT-I significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and decreased myeloperoxidase levels early in infection. Studies have shown that AT-I may reduce the levels of IL-1ß and IL-10 by inhibiting the MyD88/ NF-κB pathway. The drug combined with natamycin can increase corneal transparency in infected mice. CONCLUSION: AT-I may inhibit MyD88 / NF-κB pathway and the secretion of inflammatory factors IL-1 ß and IL-10 to achieve the therapeutic effect of fungal keratitis.

Human Corneal Epithelial Cells Internalize Aspergillus flavus Spores by Actin-Mediated Endocytosis.

Human corneal epithelial (HCE) cells play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of Aspergillus flavus conidia by RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidium-containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D inhibited the actin-mediated endocytosis of the conidia. Immunolabeling of the early endosomal markers CD71 and early endosomal antigen (EEA1) and the late endosomal markers lysosome-associated membrane protein 1 (LAMP1), Rab7, and cathepsin G showed that endosomal proteins were recruited to the site of conidia and showed maturation of the conidium-containing phagosomes. Lysotracker red DND 99 labeling showed the acidification of the phagosomes containing conidia. Phagosome-specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidium-containing phagosomes. These results show that the ocular surface epithelium contributes actively to antifungal defense by the phagocytosis of invading fungal conidia.

Thymic stromal lymphopoietin participates in the TLR2-and TLR4-dependent immune response triggered by Aspergillus fumigatus in human corneal cells.

Fungal keratitis constitutes a serious vision-threatening disease. Toll-like receptors (TLRs) comprise key mediators of innate immunity triggered by Aspergillus fumigatus (AF) in the cornea, but the messenger between innate and adaptive immunity remained unknown. Thymic stromal lymphopoietin (TSLP) represents a critical factor of adaptive immunity. Here we investigated the expression of TSLP in corneal epithelial and stromal cells challenged by AF and its relationship with TLRs. We stimulated corneal cells with TLR ligands zymosan or lipopolysaccharide (LPS), human recombinant TSLP, or AF hyphae for various periods, with or without prior TLR2, TLR4, or TSLP inhibition. TLR2, TLR4, TSLP, IL-8, and TNF-α release and expression were measured via enzyme-linked immunosorbent analysis, quantitative polymerase chain reaction, or western blot. Corneal cell stimulation with zymosan or LPS induced up-regulated TSLP expression. Enhanced TSLP expression was associated with AF treatment in human corneal cells; TLR2 or TLR4 inhibition impaired the AF-induced TSLP levels. Human recombinant TSLP augmented TLR2 and TLR4 expression; RNA interference of TSLP attenuated TLR, IL-8, and TNF-α expression stimulated by AF hyphae. These findings indicated that TSLP participates in the immune response of corneal cells triggered by AF, which is closely related to TLR function, and the innate immunity mediated by TLRs could be enhanced by TSLP. Innate immunity may therefore transmit inflammatory signals to adaptive immunity through activation of TSLP; in turn, adaptive immunity likely exerts certain regulatory effects on innate immunity via TSLP. That is, TSLP could interact with innate immunity mediated by TLR2 and TLR4 in human corneal cells challenged by AF and thus may serve as a messenger between the innate and adaptive immune responses in AF keratitis.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis.

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and IFN-ß. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

GSDMD, an executor of pyroptosis, is involved in IL-1ß secretion in Aspergillus fumigatus keratitis.

The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1ß was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1ß secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1ß and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.

Cationic Polyelectrolyte Nanocapsules of Moxifloxacin for Microbial Keratitis Therapy: Development, Characterization, and Pharmacodynamic Study.

Microbial keratitis (MK) is a vision-threatening disease and the fourth leading cause of blindness worldwide. In this work, we aim to develop moxifloxacin (MXN)-loaded chitosan-based cationic mucoadhesive polyelectrolyte nanocapsules (PENs) for the effective treatment of MK. PENs were formulated by polyelectrolyte complex coacervation method and characterized for their particle size, surface charge, morphology, mucoadhesive property, in-vitro and ex-vivo release, ocular tolerance, and antimicrobial efficacy studies. The pharmacodynamic study was conducted on rabbit eye model of induced keratitis and it is compared with marketed formulation (MF). Developed PENs showed the size range from 230.7 ± 0.64 to 249.0 ± 0.49 nm and positive surface charge, spherical shape along with appropriate physico-chemical parameters. Both in-vitro and ex-vivo examination concludes that PENs having more efficiency in sustained release of MXN compared to MF. Ocular irritation studies demonstrated that no corneal damage or ocular irritation. The in-vivo study proved that the anti-bacterial efficacy of PENs was improved when compared with MF. These results suggested that PENs are a feasible choice for MK therapy because of their ability to enhance ocular retention of loaded MXN through interaction with the corneal surface of the mucous membrane.

Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Resolvin D1 attenuates the inflammatory process in mouse model of LPS-induced keratitis.

The aim of this study was to investigate the effects of the lipid mediator Resolvin D1 in experimental keratitis. C57BL/6J mice were injected with lipopolysaccharide (2 µg/eye), and after 24 hours, the corneal damage was assessed. Clinical score was quantified, and corneal inflammatory biomarkers were detected by immunohistochemistry. A robust accumulation of sub-epithelial macrophages and polymorphonuclear leucocytes, chemokine (C-X-C motif) ligand 1 (also known as keratinocyte-derived chemokine), interleukin-10 and promoters of apoptosis was also observed in lipopolysaccharide-treated mice. Formyl peptide receptor 2 corneal expression was also assessed. The corneal stroma treated with lipopolysaccharide was characterized by presence of macrophages of M1-like subtype and immature fibroblastic cells, marked with Ki67, not fully differentiated in fibroblasts. Indeed, the staining of the cornea with anti-vimentin antibodies, a marker of differentiated myofibroblasts, was very faint. Resolvin D1 attenuated all the inflammatory parameters assessed in the present study, except for IL-10. In conclusion, the data presented here seem to be consistent with the hypothesis that Resolvin D1 protected the cornea from the lipopolysaccharide-induced keratitis by acting on several inflammatory components of this damage, pivoted by Formyl peptide receptor 2 (FPR2) activation and macrophages-leucocytes activity.

Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil-derived acidic mammalian chitinase.

Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase-/- mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil-derived AMCase as an essential mediator of host defense.