Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Intravital Imaging of the Human Cornea Reveals the Differential Effects of Season on Innate and Adaptive Immune Cell Morphodynamics.

PURPOSE: Defining how the in vivo immune status of peripheral tissues is shaped by the external environment has remained a technical challenge. We recently developed Functional in vivo confocal microscopy (Fun-IVCM) for dynamic, longitudinal imaging of corneal immune cells in living humans. This study investigated the effect of seasonal-driven environmental factors on the morphodynamic features of human corneal immune cell subsets. DESIGN: Longitudinal, observational clinical study. PARTICIPANTS: Sixteen healthy participants (aged 18-40 years) attended 2 visits in distinct seasons in Melbourne, Australia (Visit 1, November-December 2021 [spring-summer]; Visit 2, April-June 2022 [autumn-winter]). METHODS: Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg Engineering). Corneal scans were acquired at 5.5 ± 1.5-minute intervals for up to 5 time points. Time-lapse Fun-IVCM videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using a multiplex bead-based immunoassay. MAIN OUTCOME MEASURES: Difference in the density, morphology, and dynamic parameters of corneal immune cell subsets over the study periods. RESULTS: Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels compared with Visit 2. Clinical ocular surface parameters and the density of immune cell subsets were similar across visits. At Visit 1 , corneal epithelial DCs were larger, with a lower dendrite probing speed (0.38 ± 0.21 vs. 0.68 ± 0.33 µm/min; P < 0.001) relative to Visit 2; stromal macrophages were more circular and had less dynamic activity (Visit 1, 7.2 ± 1.9 vs. Visit 2, 10.3 ± 3.7 dancing index; P < 0.001). Corneal T cell morphodynamics were unchanged across periods. Basal tear levels of interleukin 2 and CXCL10 were relatively lower during spring-summer. CONCLUSIONS: This study identifies that the in vivo morphodynamics of innate corneal immune cells (DCs, macrophages) are modified by environmental factors, but such effects are not evident for adaptive immune cells (T cells). The cornea is a potential in vivo window to investigate season-dependent environmental influences on the human immune system. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The effect of topical decorin on temporal changes to corneal immune cells after epithelial abrasion.

BACKGROUND: Corneal immune cells interact with corneal sensory nerves during both homeostasis and inflammation. This study sought to evaluate temporal changes to corneal immune cell density in a mouse model of epithelial abrasion and nerve injury, and to investigate the immunomodulatory effects of topical decorin, which we have shown previously to promote corneal nerve regeneration. METHODS: Bilateral corneal epithelial abrasions (2 mm) were performed on C57BL/6J mice. Topical decorin or saline eye drops were applied three times daily for 12 h, 24 h, 3 days or 5 days. Optical coherence tomography imaging was performed to measure the abrasion area. The densities of corneal sensory nerves (ß-tubulin III) and immune cells, including dendritic cells (DCs; CD11c+), macrophages (Iba-1+) and neutrophils (NIMP-R14+) were measured. Cx3cr1gfp/gfp mice that spontaneously lack resident corneal intraepithelial DCs were used to investigate the specific contribution of epithelial DCs. Neuropeptide and cytokine gene expression was evaluated using qRT-PCR at 12 h post-injury. RESULTS: In decorin-treated corneas, higher intraepithelial DC densities and lower neutrophil densities were observed at 24 h after injury, compared to saline controls. At 12 h post-injury, topical decorin application was associated with greater re-epithelialisation. At 5 days post-injury, corneal stromal macrophage density in the decorin-treated and contralateral eyes was lower, and nerve density was higher, compared to eyes treated with saline only. Lower expression of transforming growth factor beta (TGF-ß) and higher expression of CSPG4 mRNA was detected in corneas treated with topical decorin. There was no difference in corneal neutrophil density in Cx3cr1gfp/gfp mice treated with or without decorin at 12 h. CONCLUSIONS: Topical decorin regulates immune cell dynamics after corneal injury, by inhibiting neutrophils and recruiting intraepithelial DCs during the acute phase (< 24 h), and inhibiting macrophage density at the study endpoint (5 days). These immunomodulatory effects were associated with faster re-epithelialisation and likely contribute to promoting sensory nerve regeneration. The findings suggest a potential interaction between DCs and neutrophils with topical decorin treatment, as the decorin-induced neutrophil inhibition was absent in Cx3cr1gfp/gfp mice that lack corneal epithelial DCs. TGF-ß and CSPG4 proteoglycan likely regulate decorin-mediated innate immune cell responses and nerve regeneration after injury.

Topographical Distribution and Phenotype of Resident Meibomian Gland Orifice Immune Cells (MOICs) in Mice and the Effects of Topical Benzalkonium Chloride (BAK).

Meibomian gland orifices (MGOs) are located along the eyelid margin and secrete meibum into the tear film. The profile of resident innate immune cells (ICs) at this site is not well understood. The distribution and phenotype of resident ICs around MGOs in mice was investigated and herein defined as MGO-associated immune cells (MOICs). The effect of topical 0.1% benzalkonium chloride (BAK) on MOICs was also assessed. Eyelids from healthy CD11ceYFP and Cx3cr1gfp/gfp mice aged three or seven months were compared. ICs were identified as CD11c+, Cx3cr1+, and MHC-II+ using four-colour immunostaining and confocal microscopy. MOIC density was variable but clustered around MGOs. There were more CD11c+ MOICs in three-month-old compared with seven-month-old mice (three-month-old: 893 ± 449 cells/mm2 vs. seven-month-old: 593 ± 493 cells/mm2, p = 0.004). Along the eyelid margin, there was a decreasing gradient of CD11c+ MOIC density in three-month-old mice (nasal: 1003 ± 369 cells/mm2, vs. central: 946 ± 574 cells/mm2, vs. temporal: 731 ± 353 cells/mm2, p = 0.044). Cx3cr1-deficient mice had two-fold fewer MHC-II+ MOICs, suggesting a role for Cx3cr1 receptor signaling in meibomian gland surveillance. CD11c+ MOIC density was lower in BAK-exposed eyes compared to saline-treated controls, suggesting a change in homeostasis. This study provides novel insight into resident ICs located at MGOs, and their contribution to MG homeostasis.

Novel alterations in corneal neuroimmune phenotypes in mice with central nervous system tauopathy.

BACKGROUND: Tauopathy in the central nervous system (CNS) is a histopathological hallmark of frontotemporal dementia (FTD) and Alzheimer's disease (AD). Although AD is accompanied by various ocular changes, the effects of tauopathy on the integrity of the cornea, which is densely innervated by the peripheral nervous system and is populated by resident dendritic cells, is still unknown. The aim of this study was to investigate if neuroimmune interactions in the cornea are affected by CNS tauopathy. METHODS: Corneas from wild type (WT) and transgenic rTg4510 mice that express the P301L tau mutation were examined at 2, 6, 8, and 11 months. Clinical assessment of the anterior segment of the eye was performed using spectral domain optical coherence tomography. The density of the corneal epithelial sensory nerves and the number and field area of resident epithelial dendritic cells were assessed using immunofluorescence. The immunological activation state of corneal and splenic dendritic cells was examined using flow cytometry and compared between the two genotypes at 9 months of age. RESULTS: Compared to age-matched WT mice, rTg4510 mice had a significantly lower density of corneal nerve axons at both 8 and 11 months of age. Corneal nerves in rTg4510 mice also displayed a higher percentage of beaded nerve axons and a lower density of epithelial dendritic cells compared to WT mice. From 6 months of age, the size of the corneal dendritic cells was significantly smaller in rTg4510 compared to WT mice. Phenotypic characterization by flow cytometry demonstrated an activated state of dendritic cells (CD86+ and CD45+ CD11b+CD11c+) in the corneas of rTg4510 compared to WT mice, with no distinct changes in the spleen monocytes/dendritic cells. At 2 months of age, there were no significant differences in the neural or immune structures between the two genotypes. CONCLUSIONS: Corneal sensory nerves and epithelial dendritic cells were altered in the rTg4510 mouse model of tauopathy, with temporal changes observed with aging. The activation of corneal dendritic cells prior to the gradual loss of neighboring sensory nerves suggests an early involvement of corneal immune cells in tau-associated pathology originating in the CNS.

The neuroregenerative effects of topical decorin on the injured mouse cornea.

BACKGROUND: The cornea is innervated with a rich supply of sensory nerves that play important roles in ocular surface health. Any injury or pathology of the corneal nerves increases the risk of dry eye disease and infection. This study aims to evaluate the therapeutic potential of topical decorin to improve corneal nerve regeneration in a mouse model of sterile epithelial abrasion injury. METHODS: Bilateral central corneal epithelial abrasions (2-mm, Alger Brush) were performed on young C57BL/6 J mice to remove the corneal sensory nerves. Decorin, or vehicle, was applied topically, three times per day for 1 week or every 2 h for 6 h. Spectral-domain optical coherence tomography was performed to measure the abrasion area and corneal thickness. Wholemount immunofluorescence staining was used to assess sensory nerve regeneration (ß-tubulin III) and immune cell density (CD45, Iba1, CD11c). To investigate the specific role of dendritic cells (DCs), Cx3cr1gfp/gfp mice, which spontaneously lack resident corneal epithelial DCs, were also investigated. The effect of prophylactic topical administration of recombinant human decorin (applied prior to the abrasion) was also investigated. Nerve tracing (NeuronJ software) was performed to compare recovery of basal nerve axons and superficial nerve terminals in the central and peripheral cornea. RESULTS: At 6 h after injury, topical decorin application was associated with greater intraepithelial DC recruitment but no change in re-epithelialisation or corneal thickness, compared to the vehicle control. One week after injury, sub-basal nerve plexus and superficial nerve terminal density were significantly higher in the central cornea in the decorin-treated eyes. The density of corneal stromal macrophages in the decorin-treated eyes and their contralateral eyes was significantly lower compared to saline-treated corneas. No significant improvement in corneal nerve regeneration was observed in Cx3cr1gfp/gfp mice treated with decorin. CONCLUSIONS: Decorin promotes corneal epithelial nerve regeneration after injury. The neuroregenerative effect of topical decorin was associated with a higher corneal DC density during the acute phase, and fewer macrophages at the study endpoint. The corneal neuroregenerative effects of decorin were absent in mice lacking intraepithelial DCs. Together, these findings support a role for decorin in DC-mediated neuroregeneration following corneal abrasion injury.

Targeted delivery of mitomycin C-loaded and LDL-conjugated mesoporous silica nanoparticles for inhibiting the proliferation of pterygium subconjunctival fibroblasts.

Pterygium is a degenerative disease that characterized by excessive fibrovascular proliferation. To reduce the recurrence rate, surgery is the main strategy, in combination with adjacent procedures or adjunctive therapy. One of the most common adjunctive agents, mitomycin C (MMC), is known as an alkylating agent that inhibits fibroblast proliferation but is limitedly applied in pterygium due to various complications. A previous study demonstrated that activated pterygium subconjunctival fibroblasts overexpressed low-density lipoprotein (LDL) receptors. In this study, we designed and synthesized MMC-loaded mesoporous silica nanoparticles conjugated with LDL (MMC@MSNs-LDL) to deliver MMC into activated pterygium fibroblasts in a targeted manner. The MMC loading efficiency was approximately 6%. The cell viability test (CCK-8 assay) revealed no cytotoxicity for the empty carrier MSNs at a concentration of ≤1 mg/ml after administration for 48 h in subconjunctival fibroblasts. Primary pterygium and normal human subconjunctival fibroblasts with or without stimulation by vascular endothelial growth factor (VEGF) were treated as follows: 1) 10 µg/ml MMC@MSNs-LDL for 24 h (MMC concentration: 0.6 µg/ml); 2) 0.2 mg/ml MMC for 5 min then cultured for 24 h after MMC removal; and 3) normal culture without any drug treatment. At 24 h, the anti-proliferative effect of MMC@MSNs-LDL in activated pterygium fibroblasts was similar to that of MMC (cell viability: 46.2 ± 5.5% vs 40.5 ± 1.1%, respectively, P = 0.349). Furthermore, the cytotoxicity of MMC@MSNs-LDL to normal fibroblasts with or without VEGF stimulation was significantly lower than that of traditional MMC (cell viability: 75.6 ± 4.4% vs 36.0 ± 1.5%, respectively, P < 0.001; 84.7 ± 5.5% vs 35.7 ± 1.3%, P < 0.001). The binding of fluorescently labeled MMC@MSNs-LDL in fibroblasts was assessed using confocal fluorescence microscopy. The uptake of targeted nanoparticles in fibroblasts was time dependent and saturated at 6 h. VEGF-activated pterygium fibroblasts showed more uptake of MMC@MSNs-LDL than normal fibroblasts with or without VEGF activation (both P < 0.001). Our data strongly suggest that MMC@MSNs-LDL had an effective antiproliferative role in activated pterygium fibroblasts, with reduced toxicity to normal fibroblasts compared to traditional application of MMC. LDL-mediated drug delivery might have great potential in the management of pterygium recurrence.

Evaluation of the ocular surface characteristics and Demodex infestation in paediatric and adult blepharokeratoconjunctivitis.

BACKGROUND: To evaluate the ocular surface characteristics and the infestation of Demodex in Chinese paediatric and adult blepharokeratoconjunctivitis (BKC). METHODS: Fifty consecutive patients with BKC and 50 age- and sex-matched healthy subjects were enrolled. Lid margin characteristics and corneal disorders were evaluated under slit-lamp illumination. Four eyelashes were collected from each eye to examine Demodex infestation by light microscopy. RESULTS: Corneal neovascularization (P = 0.001) and scarring (P = 0.040) were significantly worse in children than in adults with BKC, whereas meibum quality was worse in adults (P = 0.008). Diagnosis delay was longer in children with BKC than in adults (2.2 vs 1.2 years, P = 0.022). Demodex infestation was more frequent in subjects with BKC than in healthy subjects (56% vs 26%, P = 0.002). The lid margin inflammation and meibomian gland dysfunction were worse in Demodex-positive subjects than in Demodex-negative subjects with BKC. CONCLUSIONS: Children with BKC had severer corneal disorders compared with adult BKC patients, which may be caused by a long-delayed diagnosis. Ocular demodicosis was more common in subjects with BKC. Ocular Demodex infestation was associated with worse lid margin inflammation and meibomian gland dysfunction.

Case Report: In Vivo Confocal Microscopic Appearance of Corneal Argyrosis.

SIGNIFICANCE: This report details the natural history of corneal argyrosis over 3 years using in vivo confocal microscopy to document regression of hyperreflective deposits, as well as effects on corneal nerves and endothelial cell morphology. PURPOSE: To report the in vivo confocal microscopic features and clinical characteristics of a case of bilateral corneal argyrosis. CASE REPORT: A 52-year-old man referred to us 3 months following cautery of the palpebral conjunctiva of both eyes with a silver nitrate stick was observed over the course of 3 years, during which slit-lamp photography and in vivo confocal microscopy were performed. At the first visit, slit-lamp examination showed a light blue-green discoloration and a thick, yellow, oval discoloration in the right and left cornea, respectively. One year later, under slit-lamp examination, the right cornea appeared nearly transparent, and the discoloration in the left cornea had remarkably regressed. In vivo confocal microscopy done at that time showed highly reflective deposits in Descemet membrane of the right cornea and throughout Bowman layer, the stroma, and Descemet membrane of the left cornea. Three years later, no accumulation of silver was observed during slit-lamp examination of either eye. In vivo confocal microscopy of the right cornea did not reveal any silver deposits, and the corneal structure appeared normal. In the left cornea, some silver deposits were still evident in Descemet membrane, and alterations of corneal nerve and endothelial cell morphology were also evident. CONCLUSIONS: This report reviews the 3-year natural history of a patient with corneal argyrosis. In vivo confocal microscopy demonstrates that over time the corneal argyrosis gradually resolves without any treatment. However, the presence of silver in the cornea may impact the corneal nerves and endothelial cells.