Human Corneal Epithelial Cells Research Articles

Rational design and synthesis of lumican stapled peptides for promoting corneal wound healing

PurposeLumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13C-A) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13C-A with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential. MethodsDifferent peptides staples were rationalized using LumC13C-A sequence by computational chemistry, docked to TGFβRI and the interface binding energies compared. Lowest scoring peptides were synthesized, and the toxicity of peptides tested using CCK8-based cell viability assay. The efficacy of the stapled peptides at promoting corneal wound healing was tested using a proliferation assay, an in vitro scratch assay using human corneal epithelial cells and an in vivo murine corneal debridement wound healing model. ResultsBinding free energies were calculated using MMGBSA algorithm, and peptides LumC13C and LumC13S5 displayed superior binding to ALK5 compared to the non-stapled peptide LumC13C-A. The presence of the hydrocarbon staple in LumC13C enhances the stability of the α-helical conformation, thereby facilitating more optimal interactions with the ALK5 receptor. The stapled peptides do not present cytotoxic effects on human corneal epithelial cells at a 300 nM concentration. Similar to lumican and LumC13C-A, both C13C and LumC13S5 significantly promote corneal wound healing both in vitro and in vivo. ConclusionsHighly stable and non-toxic stapled peptides designed based on LumC13, significantly promote corneal wound healing. As a proof of principle, our data shows that more stable and pharmacologically relevant peptides can be designed based on endogenous peptide sequences for treating various corneal pathologies.

Development of Osthole-Loaded Microemulsions as a Prospective Ocular Delivery System for the Treatment of Corneal Neovascularization: In Vitro and In Vivo Assessments.

Osthole (OST), a natural coumarin compound, has shown a significant inhibitory effect on corneal neovascularization (CNV). But, its effect on treating CNV is restricted by its water insolubility. To overcome this limitation, an OST-loaded microemulsion (OST-ME) was created to improve the drug's therapeutic effect on CNV after topical administration. The OST-ME formulation comprised Capryol-90 (CP-90), Cremophor® EL (EL-35), Transcutol-P (TSP) and water, and sodium hyaluronate (SH) was also included to increase viscosity. The OST-ME had a droplet size of 16.18 ± 0.02 nm and a low polydispersity index (0.09 ± 0.00). In vitro drug release from OST-ME fitted well to the Higuchi release kinetics model. Cytotoxicity assays demonstrated that OST-ME was not notably toxic to human corneal epithelial cells (HCECs), and the formulation had no irritation to rabbit eyes. Ocular pharmacokinetics studies showed that the areas under the concentration-time curves (AUC0-t) in the cornea and conjunctiva were 19.74 and 63.96 μg/g*min after the administration of OST-ME, both of which were 28.2- and 102.34-fold higher than those after the administration of OST suspension (OST-Susp). Moreover, OST-ME (0.1%) presented a similar therapeutic effect to commercially available dexamethasone eye drops (0.025%) on CNV in mouse models. In conclusion, the optimized OST-ME exhibited good tolerance and enhanced 28.2- and 102.34-fold bioavailability in the cornea and conjunctiva tissues compared with suspensions in rabbit eyes. The OST-ME is a potential ocular drug delivery for anti-CNV.

The cGAS-STING pathway-dependent sensing of mitochondrial DNA mediates ocular surface inflammation

The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models—a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples—along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)—stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.

Identification and Analysis of Primary Cilia in the Corneal Endothelial Cells of Patients with Bullous Keratopathy

Purpose To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK). Methods This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68–90 years) with BK who underwent Descemet’s stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin. Results Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients. Conclusion The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK.

Reducing Sialylation Enhances Electrotaxis of Corneal Epithelial Cells.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

Berberine ameliorate inflammation and apoptosis via modulating PI3K/AKT/NFκB and MAPK pathway on dry eye

BackgroundDry eye disease (DED) is a multifactorial disease in ocular surface, and inflammation plays an etiological role. Berberine (BBR) has shown efficacy in treating inflammatory diseases. Yet, there was no adequate information related to the therapeutic effects of BBR for DED. PurposeTo detect the effects and explore the potential mechanisms of BBR on DED. Study DesignIn vitro, in vivo study and network pharmacology analysis were involved. MethodThe human corneal epithelium cells viability was evaluated with different concentrations of BBR. Dry eye murine model was established by exposing to the desiccating stress, and Ciclosporin (CSA), BBR eye drops or vehicle were topical administration for 7 days. The phenol red cotton tests, Oregon-green-dextran staining and Periodic acid-Schiff staining were performed and evaluated the dry eye after treatment. Inflammation and apoptosis levels of ocular surface were quantified. The potential targets related to berberine and dry eye were collected from databases. The Protein-Protein interaction network analysis and GO & KEGG enrichment analysis were realized by STRING database, Metascape platform and Cytoscape software to find core targets and signaling pathways. The SchrÖdinger software was used to molecular docking and PyMOL software to visualization. Finally, the levels of PI3K/AKT/NFκB and MAPK pathways were detected. ResultThe data revealed BBR could rescue impaired HCE under hyperosmotic conditions. In addition, BBR eye drops could ameliorate dry eye. And BBR eye drops suppressed the inflammatory factors and CD4+T cells infiltration in conjunctiva. Besides, BBR eye drops protected ocular surface by avoiding the severe apoptosis and decreasing the level of MMP-3 and MMP-9. 148 common targets intersection between BBR and dry eye were found via network pharmacology analysis. Core proteins and core pathways were identified through PPI and GO&KEGG enrichment analysis. Molecular docking displayed excellent binding between BBR and those core targets. Finally, in vivo study verified that BBR eye drops had a therapeutic effect in dry eye by inhibiting PI3K/AKT/NFκB and MAPK pathways. ConclusionThe research provided convincing evidence that BBR could be a candidate drug for dry eye.

The Biocompatibility Analysis of Artificial Mucin-Like Glycopolymers.

The ocular surface is covered by a tear film consisting of an aqueous/mucin phase and a superficial lipid layer. Mucins, highly O-glycosylated proteins, are responsible for lubrication and ocular surface protection. Due to contact lens wear or eye disorders, lubrication of the ocular surface can be affected. Artificial glycopolymers which mimic natural mucins could be efficient in ophthalmic therapy. Various neutral, positively, and negatively charged mucin-mimicking glycopolymers were synthesized (n = 11), cultured in different concentrations (1%, 0.1%, and 0.01% w/v) with human corneal epithelial cells (HCE), and analyzed by various cytotoxicity/viability, morphology, and immunohistochemistry (IHC) assays. Six of the eleven glycopolymers were selected for further analysis after cytotoxicity/viability assays. We showed that the six selected glycopolymers had no cytotoxic effect on HCE cells in the 0.01% w/v concentration. They did not negatively affect cell viability and displayed both morphology and characteristic markers as untreated control cells. These polymers could be used in the future as mucin-mimicking semi-synthetic materials for lubrication and protection of the ocular surface.

Selective effects of estradiol on human corneal endothelial cells

In Fuchs endothelial corneal dystrophy (FECD), mitochondrial and oxidative stresses in corneal endothelial cells (HCEnCs) contribute to cell demise and disease progression. FECD is more common in women than men, but the basis for this observation is poorly understood. To understand the sex disparity in FECD prevalence, we studied the effects of the sex hormone 17-β estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of HCEnCs grown under physiologic ([O2]2.5) and hyperoxic ([O2]A) conditions. We hypothesized that E2 would counter the damage of oxidative stress generated at [O2]A. HCEnCs were treated with or without E2 (10 nM) for 7–10 days under both conditions. Treatment with E2 did not significantly alter HCEnC density, viability, ROS levels, oxidative DNA damage, oxygen consumption rates, or extracellular acidification rates in either condition. E2 disrupted mitochondrial morphology in HCEnCs solely from female donors in the [O2]A condition. ATP levels were significantly higher at [O2]2.5 than at [O2]A in HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the resilience of HCEnCs against hyperoxic stress. The effects of hyperoxia and E2 on HCEnCs from female donors suggest cell sex-specific mechanisms of toxicity and hormonal influences.

Development of mucoadhesive Timolol loaded chitosan-nanocomposite to treat glaucoma

Timolol Maleate is an aqueous soluble β-blocker antiglaucoma drug used to suppress intraocular pressure. Several commercially available ocular formulations are not effective in delivering to the target site due to their water-soluble property and low mucoadhesiveness. Hence, there is a requirement for a highly mucoadhesive drug-loaded nanocomposite to suppress intraocular pressure with enhanced bioavailability. Herein, we have prepared a mucoadhesive Timolol-loaded graphene quantum dot-chitosan-nanocomposite to treat glaucoma in response to lysozyme, secreted in the tear fluid. The as-prepared nanocomposite has been characterized through high resolution-transmission electron microscopic, X-ray photoelectron spectroscopic, X-ray diffraction, and Fourier transform infrared spectral studies. The nanocomposite showed 93.74 % encapsulation efficiency with a loading capacity of 7.73 %. Further, 89.26 %, 95.62 %, and 99.29 % of drug release were observed from the nanocomposite in the presence of 1, 1.5, and 2 mg/mL of lysozyme. The mucoadhesion property has been confirmed by the increment in the particle size, fluorescence spectral variations, and Fourier transform infrared spectroscopic studies in the presence of mucin nanoparticles of size 291 nm. Interestingly, mucoadhesion has been demonstrated by pointing to the quenching in the luminescence of mucin. Further, in vitro biocompatibility assay on human corneal epithelial cells showed ≥80 % cell viability. Hence, this study offers the utilization of naturally secreting enzymes for drug delivery applications instead of uncontrolled pH and temperature-triggered releases.

ROS-Mediated Fragmentation Alters the Effects of Hyaluronan on Corneal Epithelial Wound Healing.

A buildup of reactive oxygen species (ROS) occurs in virtually all pathological conditions. Hyaluronan (HA) is a major extracellular matrix component and is susceptible to oxidation by reactive oxygen species (ROS), yet the precise chemical structures of oxidized HA products (oxHA) and their physiological properties remain largely unknown. This study characterized the molecular weight (MW), structures, and physiological properties of oxHA. For this, high-molecular-weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl). ROS lead to the fragmentation of HA, with the oxHA products produced by HOCl exhibiting an altered chemical structure while those produced by H2O2 do not. HMWHA promotes the viability of human corneal epithelial cells (hTCEpi), while low MWHA (LMWHA), ultra-LMWHA (ULMWHA), and most forms of oxHA do not. HMWHA and LMWHA promote hTCEpi proliferation, while ULMWHA and all forms of oxHA do not. LMWHA and some forms of oxHA promote hTCEpi migration, while HMWHA does not. Finally, all native forms of HA and oxHA produced by HOCl promote in vivo corneal wound healing, while oxHA produced by H2O2 does not. Taken together, our results show that HA fragmentation by ROS can alter the physiological activity of HA by altering its MW and structure.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. In vitroresults showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium.In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p= 0.046), corneal fluorescein staining (p= 0.021), and International oGVHD Consensus Group severity scores (p= 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the HGF-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE), and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (DKO cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude and duration of c-Met receptor signaling via immunoblot, and receptor trafficking by immunofluorescence. Single knockout cells displayed a modest decrease in receptor ubiquitylation and a slight increase in phosphorylation as compared to control. DKO cells did not have detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live cell timelapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are two promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Cis-urocanic acid improves cell viability and suppresses inflammasome activation in human retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a common eye disease among the elderly, which can result in impaired vision and irreversible loss of vision. The majority of patients suffer from the dry (also known as the atrophic) form of the disease, which is completely lacking an effective treatment. In the present study, we evaluated the potential of cis-urocanic acid (cis-UCA) to protect human ARPE-19 cells from cell damage and inflammasome activation induced by UVB light. Urocanic acid is a molecule normally present in human epidermis. Its cis-form has recently been found to alleviate UVB-induced inflammasome activation in human corneal epithelial cells. Here, we observed that cis-UCA is well-tolerated also by human retinal pigment epithelial (RPE) cells at a concentration of 100 μg/ml. Moreover, cis-UCA was cytoprotective and efficiently diminished the levels of mature IL-1β, IL-18, and cleaved caspase-1 in UVB-irradiated ARPE-19 cells. Interestingly, cis-UCA also reduced DNA damage, whereas its effect against ROS production was negligible. Collectively, cis-UCA protected ARPE-19 cells from UVB-induced phototoxicity and inflammasome activation. This study indicates that due to its beneficial properties of preserving cell viability and preventing inflammation, cis-UCA has potential in drug development of chronic ocular diseases, such as AMD.

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread.

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure-activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread

AbstractHerpes simplex virus (HSV‐1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late‐stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV‐1. A range of HS‐oligosaccharides was prepared to examine the importance of chain length and 2‐O‐sulfation of iduronic moieties for Hpse inhibition. It was found that hexa‐ and octasaccharides potently inhibited the enzyme and that 2‐O‐sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure–activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV‐1 with the hexa‐ and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.

The Evolving Therapeutics of Endothelial Disease

Abstract: Endothelial dysfunction is one of the leading indications for corneal transplantation. Globally, there is a shortage of donor corneas, which is partly because of the lack of eye banking resources in emerging countries. Given this global shortage, there is naturally heightened interest surrounding pharmacological agents, genetic therapy, and endothelial cell–based therapeutic modalities. Endothelial cells have limited mitotic capacity in vivo, and thus, studies have been conducted for their ex vivo expansion using mitogens. Similarly, pluripotent stem cells have been used to differentiate into human corneal endothelial cells with varying degrees of success. Different strategies have also been developed for injecting and delivering these cells into the human eye. This review aims to compile the information published on the latest advances in endothelial disease therapy.

The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity

The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Prospective, comparative, observational study. A total of 55 patients with BK and 31 patients with cataract (Cat) as control. A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n=18) and Cat (n=8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n=55) and Cat (total n=31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P=0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P=0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Proprietary or commercial disclosure may be found after the references.

EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption.

Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by Pseudomonas aeruginosa, specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects exotoxin A (EXOA), a ribotoxin released by P. aeruginosa type 2 secretion system (T2SS), during chronic infection. Mechanistically, EXOA-driven eukaryotic elongation factor 2 (EEF2) ribosylation and covalent inactivation promote ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, diphtheria toxin and cholix toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAKα- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, cystic fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2.

Dermatophagoides Pteronyssinus 1 (DerP1) May Trigger NLRP3-Mediated Corneal Epithelial Cell Pyroptosis by Elevating Interleukin-33 Expression Levels

Purpose To characterize the in vivo effects of Dermatophagoides pteronyssinus 1 (DerP1) in mice and determine the underlying NLRP3 inflammasome-mediated pyroptosis signaling mechanisms in the human corneal epithelial cells (HCECs). Methods DerP1 was used to induce allergic conjunctivitis in C57 mice. HCECs were sensitized with DerP1 in vitro to mimic their condition observed in allergic conjunctivitis in vivo. Transmission electron microscopy was used to evaluate pyroptosis in the HCECs, enzyme-linked immunosorbent assays to assess interleukin (IL)-33, IL-1β and IL-4 levels, flow cytometry to detect the proportion of Th2 cells, MTT assays to assess cell metabolic activity, immunofluorescence to evaluate the effects of DerP1 on functional HCEC phenotypes, and Western blot assays to detect the expression of NOD-like receptor family pyrin domain-containing 3 (NLRP3), gasdermin D (GSDMD), N-terminal fragment of GSDMD (GSDMD-N), pro-caspase-1, cleaved caspase-1, IL-1β, and IL-33. IL-33 expression in the HCECs was knocked down via lentivirus transfection. Results In vivo, DerP1 promotes pyroptosis, production of Th2 inflammatory cytokines and IL-33, and NLRP3 activation in mouse corneas. In vitro, pyroptotic bodies were found in the HCECs after sensitization with DerP1. Various concentrations of DerP1 increased the expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved caspase-1, and IL-1β in the HCECs, with the largest increase observed after exposure to 20 µM DerP1. In vitro, recombinant human IL-33 mediated the expression of pyroptotic biomarkers in the HCECs, whereas IL-33 silencing diminished 20 µM DerP1-induced increase in their expression levels. Conclusions DerP1 induces pyroptosis and allergic conjunctivitis, the expression of Th2 inflammatory cytokines, NLRP3 activation, and IL-33 in mouse corneas in our model. These effects would attribute to its activating NLRP3–GSDMD signaling pathway axis via enhancing IL-33 expression in HCECs.

Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches.

Tilapia skin is a great source of collagen. Here, we aimed to isolate and identify the peptides responsible for combating dry eye disease (DED) in tilapia skin peptides (TSP). In vitro cell DED model was used to screen anti-DED peptides from TSP via Sephadex G-25 chromatography, LC/MS/MS, and in silico methods. The anti-DED activity of the screened peptide was further verified in the mice DED model. TSP was divided into five fractions (TSP-I, TSP-II, TSP-III, TSP-IV, and TSP-V), and TSP-II exerted an effective effect for anti-DED. A total of 131 peptides were identified using LC/MS/MS in TSP-II, and NGGPSGPR (NGG) was screened as a potential anti-DED fragment in TSP-II via in silico methods. In vitro, NGG restored cell viability and inhibited the expression level of Cyclooxygenase-2 (COX-2) protein in Human corneal epithelial cells (HCECs) induced by NaCl. In vivo, NGG increased tear production, decreased tear ferning score, prevented corneal epithelial thinning, alleviated conjunctival goblet cell loss, and inhibited the apoptosis of corneal epithelial cells in DED mice. Overall, NGG, as an anti-DED peptide, was successfully identified from TSP, and it may be devoted to functional food ingredients or medicine for DED.