Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia

Background Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. Methods Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. Results Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. Conclusions Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Lipopolysaccharide Enhances Genotoxicity by Activating GADD45G and NF-κB in Human Corneal Epithelial Cells

As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 μg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 μg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.

Instillation of Ophthalmic Formulation Containing Nilvadipine Nanocrystals Attenuates Lens Opacification in Shumiya Cataract Rats

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

Development of Lutein-Containing Eye Drops for the Treatment of Dry Eye Syndrome

Dry eye syndrome (DES) is a common ophthalmological disease that decreases tear secretion and causes dryness, photophobia, pain, severe corneal rupture, and even blindness. Ocular and lacrimal gland inflammation is one of the pathological mechanisms underlying DES. Therefore, effective suppression of inflammation is a crucial strategy for the treatment of DES. Lutein, commonly found in healthy foods, has anti-inflammatory effects in corneal or retina-related cells and may be a potential therapy for DES. The addition of lutein to artificial tears (AT) as an eye-drop formulation for DES treatment in a mouse model was studied in the present work. Polyvinyl alcohol (PVA) was used as a thickener to increase the viscosity of eye drops to prolong drug retention on the ocular surface. A WST-8 assay in human corneal epithelial cells (HCE-2) showed that a concentration of <5 μM lutein (L5) and <1% PVA (P1) maintained the cell viability at 80%. A real-time PCR showed that the inflamed human corneal epithelial cells (HCECs) cocultured with L5P1 had downregulated expression of inflammatory genes such as IL-1β, IL-6, and TNF-α. In a benzalkonium chloride- (BAC) induced DES mouse model, AT/L5P1 could repair damaged corneas, elevate tear secretion, increase the number of goblet cells, and inhibit the production of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in the cornea. In conclusion, we demonstrate that lutein/PVA as eye drops could prolong the drug ocular retention time and effectively to decrease inflammation in DES mice. Therefore, lutein, obtained from eye drops, has a potential therapeutic role for DES.

Purification, Characterization of an Entomopathogenic Fungal Lectin from Purpureocillium Lilacinum, and its Involvement in Pathogenesis Leading to Mycotic Keratitis

A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non reducing conditions. PCL is a blood group non specific lectin and has highest affinity towards Chitin, Mucin, asialo mucin, Fetuin with a MIC of 0.15µg/mL and also recognizes L-fucose, galactose, lactose, N-acetly galactosamine, Hyaluronic acid. PCL is stable up to 60 ºC and within the pH range 4–8. To understand its role in pathogenesis, effect of PCL was evaluated on Human Corneal Epithelial Cells (HCECs). PCL showed strong glycan mediated binding to HCECsand PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

Exploring the Effect of Structure-Based Scaffold Hopping on the Inhibition of Coxsackievirus A24v Transduction by Pentavalent N-Acetylneuraminic Acid Conjugates

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.