Key Role of Staphylococcal Fibronectin-Binding Proteins During the Initial Stage of Staphylococcus aureus Keratitis in Humans

ObjectivesStaphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis.MethodsHuman corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells.ResultsSuperficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium (p < 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin (p < 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium (p < 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt.ConclusionS. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium.

Stem Cell Niche Microenvironment: Review

The cornea comprises a pool of self-regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self-renewing and skeletal tissues, hold immense interest for tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN and artificial stem cells for self-renewal.

Effects of ambient particulate matter on a reconstructed human corneal epithelium model

We evaluated the effects of ambient particulate matter (PM) on the corneal epithelium using a reconstructed human corneal epithelium (HCE) model. We collected two PM size fractions [aerodynamic diameter smaller than 2.4 µm: PM0.3–2.4 and larger than 2.4 µm: PM>2.4] and exposed these tissues to PM concentrations of 1, 10, and 100 µg/mL for 24 h. After exposure, cell viability and interleukin (IL) IL-6 and IL-8 levels were determined, and haematoxylin and eosin and immunofluorescence staining of the zonula occludens-1 (ZO-1) were performed on tissue sections. In addition, the effects of a certified reference material of urban aerosols (UA; 100 µg/mL) were also examined as a reference. The viability of cells exposed to 100 μg/mL UA and PM>2.4 decreased to 76.2% ± 7.4 and 75.4% ± 16.1, respectively, whereas PM0.3–2.4 exposure had a limited effect on cell viability. These particles did not increase IL-6 and IL-8 levels significantly even though cell viability was decreased in 100 μg/mL UA and PM>2.4. ZO-1 expression was reduced in a dose-dependent manner in all groups. Reconstructed HCE could be used as an in vitro model to study the effects of environmental PM exposure on ocular surface cell viability and inflammation.

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.