Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

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.

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.

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.

Short chain fatty acids inhibit corneal inflammatory responses to TLR ligands via the ocular G-protein coupled receptor 43.

PURPOSE: Short chain fatty acids (SCFAs) produced by gut microbiota are known to play primary roles in gut homeostasis by immunomodulation partially through G-protein coupled receptors (GPR) 43. Using mouse models of TLR ligand induced keratitis, we investigated whether SCFAs and GPR43 play any regulatory roles in the pathogenesis of inflammatory responses in the eye. METHODS: Both human and mouse eyes were labeled with a specific antibody for GPR43 and imaged by a laser scanning confocal microscope. Corneal cups from naïve C57BL/6J (B6) and GPR43 knockout (KO) mice were stimulated with TLR ligands in the presence or absence of sodium butyrate overnight and then processed for RT-PCR assay for expression of GPR43 and cytokines. Keratitis was induced by Poly I:C in wild type (WT) B6, GPR43KO and chimeric mice and the disease severity was evaluated by the corneal fluorescein staining test, and infiltrating cell staining and calculating in corneal whole mount. RESULTS: GPR43 is expressed in both human and mouse eyes and the expression is bidirectionally regulated by TLR ligands and butyrate. Butyrate significantly inhibited inflammation caused by several TLR ligands such as Poly I:C, Flagellin, and CpG-ODN (TLR-3, 5 and 9 agonists, respectively) in WT, but not GPR43KO, mice. Butyrate inhibition of TLR-induced keratitis is mediated by the GPR43 expressed in tissue but not hematopoietic, cells. CONCLUSIONS: This is the first report to demonstrate of the protective effect of SCFAs on microbial keratitis, and the dynamic expression and anti-inflammatory function of GPR43 in the eye. SCFAs can modulate inflammation and immunity in the eye through GPR43.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Purpose: Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Methods: Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Results: Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10 days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Conclusions: Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

Endogenous TSG-6 modulates corneal inflammation following chemical injury.

PURPOSE: Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) is upregulated in various pathophysiological contexts, where it has a diverse repertoire of immunoregulatory functions. Herein, we investigated the expression and function of TSG-6 during corneal homeostasis and after injury. METHODS: Human corneas, eyeballs from BALB/c (TSG-6+/+), TSG-6+/- and TSG-6-/- mice, human immortalized corneal epithelial cells and murine corneal epithelial progenitor cells were prepared for immunostaining and real time PCR analysis of endogenous expression of TSG-6. Mice were subjected to unilateral corneal debridement or alkali burn (AB) injuries and wound healing assessed over time using fluorescein stain, in vivo confocal microscopy and histology. RESULTS: TSG-6 is endogenously expressed in the human and mouse cornea and established corneal epithelial cell lines and is upregulated after injury. A loss of TSG-6 has no structural and functional effect in the cornea during homeostasis. No differences were noted in the rate of corneal epithelial wound closure between BALB/c, TSG-6+/- and TSG-6-/- mice. TSG-6-/- mice presented decreased inflammatory response within the first 24 h of injury and accelerated corneal wound healing following AB when compared to control mice. CONCLUSION: TSG-6 is endogenously expressed in the cornea and upregulated after injury where it propagates the inflammatory response following chemical injury.

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.

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.

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.

Protective effects of resolvin D1 in Pseudomonas aeruginosa keratitis.

PURPOSE: Here, we explored the protective effects of resolvin D1 (RvD1) in Pseudomonas aeruginosa (PA) keratitis. METHODS: C57BL/6 (B6) mice were used as an animal model of PA keratitis. Plate counting and clinical scores were used to assess the severity of the infection and the therapeutic effects of RvD1 in the model. Myeloperoxidase assay was used to detect neutrophil infiltration and activity. Quantitative PCR (qPCR) was used to examine the expression of proflammatory and anti-inflammatory mediators. Immunofluorescence staining and qPCR were performed to identify macrophage polarization. RESULTS: RvD1 treatment alleviated PA keratitis severity by decreasing corneal bacterial load and inhibiting neutrophil infiltration in the mouse model. Furthermore, RvD1 treatment decreased mRNA levels of TNF-α, IFN-γ, IL-1ß, CXCL1, and S100A8/9 while increasing those of IL-1RA, IL-10, and TGF-ß1. RvD1 treatment also reduced the aggregation of M1 macrophages and increased that of M2 macrophages. RvD1 provided an auxiliary effect in gatifloxacin-treated mice with PA keratitis. CONCLUSION: Based on these findings, RvD1 may improve the prognosis of PA keratitis by inhibiting neutrophil recruitment and activity, dampening the inflammatory response, and promoting M2 macrophage polarization. Thus, RvD1 may be a potential complementary therapy for PA keratitis.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

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.

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.

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.

Developing advanced therapeutics through the study of naturally occurring immune-mediated ocular disease in domestic animals.

This review, which is part of the "Currents in One Health" series, describes the importance of the study of immune-mediated ocular disease in the development of innovative therapeutics, such as cell and gene therapy for the eye. Recent examples of cell and gene therapy studies from the author's laboratory are reviewed to emphasize the importance of One Health initiatives in developing innovative therapies for ocular diseases. Spontaneous immune-mediated corneal disease is common in horses, cats, dogs, and humans. Autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) injected subconjunctivally resulted in the resolution of naturally occurring immune-mediated keratitis (IMMK) without adverse effects. These results support that autologous subconjunctival BM-MSC therapy may be a viable treatment alternative for IMMK. Furthermore, the use of subconjunctival MSCs may be an effective method to treat ocular surface immune-mediated diseases in humans and other species, including herpetic stromal keratitis and immunologic dry eye disease. Furthermore, the use of adeno-associated viral (AAV) vectors to deliver the immunosuppressive transgene cDNA of equine interleukin 10 (eqIL-10) or human leukocyte antigen G injected intravitreally was shown to be safe and inhibited the development of uveitis in the experimental autoimmune uveitis rat model. Efficacy and safety studies of ocular gene therapy in models will pave the way for clinical trials in animals with naturally occurring immune-mediated diseases, such as a therapeutic clinical trial for AAV-eqIL-10 in horses with equine recurrent uveitis.

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.

Perillaldehyde Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting Inflammatory Cytokines and LOX-1.

PURPOSE: The purpose of this research was to explore the antifungal and anti-inflammatory effects of perillaldehyde (PAE) in Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanism. METHODS: The biofilm formation, adherence assay, and propidium iodide uptake test were used to determine the possible mechanism of PAE in terms of antifungal effects in vitro. The severity of corneal infection was evaluated by clinical scores. The immunofluorescence staining (IFS) was adopted to detect the number of macrophages in infected corneas. Draize test was performed to assess the ocular toxicity of PAE. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot reflected the expression of inflammatory cytokines and Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) in mice corneas and RAW264.7 cells. RESULTS: PAE was able to inhibit the formation of biofilm, reduce conidial adhesion, and damage the integrity of membranes to exert antifungal activity. In C57BL/6 mice models, PAE alleviated the severity of infected corneas, reduced the recruitment of macrophages and had low ocular toxicity. In addition, the mRNA and protein levels of TNF-α, CCL-2, and LOX-1 could be significantly decreased by the application of PAE after A. fumigatus infection in vivo and in vitro. CONCLUSION: Our study indicated that PAE protected against A. fumigatus keratitis by reducing fungal load, accumulation of macrophages, and inhibiting the expression of inflammatory cytokines.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation.

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis.

PURPOSE: To explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. RESULTS: CS at 400 µg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1ß, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. CONCLUSION: CS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.