Corneal Epithelial Dendritic Cells Research Articles

Intravital imaging of the human cornea reveals the differential effects of season on innate and adaptive immune cell morphodynamics

While the external environment has been shown to shape the systemic human immune landscape, defining the in vivo immune status of peripheral tissues 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 density, morphology and dynamic behavior of human corneal immune cell subsets. Longitudinal, observational clinical study. Sixteen healthy participants (18-40 years) attended two visits in distinct seasons in Melbourne, Australia (Visit 1: Spring/Summer: November-December 2021; Visit 2: Autumn/Winter: April-June 2022). Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg HRT-3, Rostock Corneal Module). Volume scans (80μm) were acquired at 5.5±1.5 minute intervals, for up to five timepoints. Time-lapse videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using multiplex bead-based immunoassay. Difference in the density, morphological and dynamic parameters of corneal immune cell subsets over the study periods. Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels than Visit 2. Clinical ocular surface parameters, and the density of immune cell subsets were similar across visits. At Visit 1 (Spring/Summer), corneal epithelial DCs were larger and more elongated, with a lower dendrite probing speed (0.38±0.21 vs 0.68±0.33μm/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). T cell morphology and dynamics were unchanged across periods. Basal tear levels of IL-2 and CXCL10 were lower during Spring/Summer. This novel study shows 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 non-invasive, in vivo 'window' to season-dependent changes to the human immune system, with capacity to yield new insight into environmental influences on immune regulation.

Corneal Epithelial Dendritic Cells: An Objective Indicator for Ocular Surface Inflammation in Patients with Obstructive Meibomian Gland Dysfunction?

ABSTRACT Purpose To examine whether corneal epithelial dendritic cells (CEDC) could serve as an indicator to distinguish obstructive meibomian gland dysfunction (MGD) with or without ocular surface inflammation (OSI). Methods We performed a case-control study on patients with diagnosed obstructive MGD between August 2017 and November 2019. Results 30 MGD cases and 25 healthy controls were recruited. The classification of MGD patients with and without OSI was based on the tear pro-inflammatory cytokine levels. Compared with the MGD without OSI and the control group, a higher CEDC density was detected in the MGD with OSI subgroup. The presence of >15.6 cells/mm2 CEDC had a sensitivity of 73% and specificity of 75% for the diagnosis of MGD with OSI. Conclusions OSI is not present in all patients with obstructive MGD. Evaluation of CEDC density in the central cornea may help identify whether MGD is concomitant with OSI.

Corneal epithelial dendritic cells, tear neuropeptides and corneal nerves continue to be affected more than 12 months after LASIK.

LASIK causes corneal nerve damage and may affect the neuro-immune crosstalk. This study examined the effects of LASIK on corneal epithelial dendritic cells (CEDC) density and morphology and explored their relationships with corneal nerves and tear neuropeptides. A grading system was developed to assess CEDC morphology. Intra- and inter-observer repeatability of the CEDC morphology grading system was established using kappa (κ). In vivo confocal microscope images of the central cornea were captured from 20 participants who had undergone LASIK 12-16 months earlier and 20 controls (age 18-32 years, 55%F). CEDC density was counted manually, and CEDC morphology was assessed using a new grading system. CEDC sub-types (contacting nerves [CEDCc] and not contacting nerves [CEDCnc]) were also assessed. Differences in CEDC density and morphology were examined using mixed models and chi-squared test. Relationships between CEDC and corneal nerve parameters and tear substance P were explored using Spearman's correlation. Excellent intra- and inter-observer repeatability was demonstrated for the grading system (κ=0.82-0.97). In post-LASIK participants, CEDC density was lower compared with controls (5 [0-34] vs. 21 [7-77] cells/mm2 ; p= 0.01), and the proportion of CEDC with thick dendrites was higher (55%-73% vs. 11%-21%, p< 0.003). Higher tear substance P levels were associated with higher CEDC density (rho=0.48, p= 0.003). Fewer nerve interconnections were observed in participants in whom CEDC had dendrites (p= 0.03). CEDC sub-types followed a similar pattern to CEDC. The findings suggest that CEDC may remain altered more than 12 months post-LASIK. The association with substance P suggests a role for CEDC in corneal neurogenic inflammation.

Altered Corneal Epithelial Dendritic Cell Morphology and Phenotype Following Acute Exposure to Hyperosmolar Saline.

PurposeThe purpose of this study was to assess the morphological and phenotypic responses of corneal epithelial dendritic cells (DCs) to acute topical hyperosmolar stress, given a pathogenic role for tear hyperosmolarity in dry eye disease (DED).MethodsC57BL/6J mice were anesthetized and received 350 mOsm/L (physiological; n = 5 mice), 450 mOsm/L (n = 6), or 600 mOsm/L (n = 6) saline on a randomly assigned eye. Corneas were harvested 2 hours later. Immunofluorescent staining was performed using CD45, CD86, and CD68 antibodies to investigate DC morphology (density, viability, field area, circularity, and dendritic complexity) and immunological phenotype. Flow cytometry was used to confirm CD86 and CD68 expression in CD11c+ DCs, using C57BL/6J mice that received topical applications of 350 mOsm/L, 450 mOsm/L, or 600 mOsm/L (n = 5 per group) bilaterally for 2 hours.ResultsFollowing exposure to 450 mOsm/L topical saline for 2 hours, DCs in the central and peripheral cornea were larger (field area: Pcentral = 0.005, Pperipheral = 0.037; circularity: Pcentral = 0.026, and Pperipheral = 0.013) and had higher expression of CD86 compared with 350 mOsm/L controls (immunofluorescence: P < 0.0001; flow cytometry: P = 0.0058). After application of 600 mOsm/L saline, DC morphology was unchanged, although the percentage of fragmented DCs, and phenotypic expression of CD86 (immunofluorescence: P < 0.0001; and flow cytometry: P = 0.003) and CD68 (immunofluorescence: P = 0.024) were higher compared to 350 mOsm/L controls.ConclusionsShort-term exposure to mild hyperosmolar saline (450 mOsm/L) induced morphological and phenotypic maturation in corneal epithelial DCs. More severe hyperosmolar insult (600 mOsm/L) for 2 hours appeared toxic to these cells. These data suggest that hyperosmolar conditions activate corneal DCs, which may have implications for understanding DC activation in DED.

The Relationship Between Corneal Nerve Density and Hemoglobin A1c in Patients With Prediabetes and Type 2 Diabetes.

PurposeDecreased corneal nerve fiber density and higher corneal epithelial dendritic cells have been reported in established patients with type 2 diabetes; however, alterations in the subbasal nerve plexus in prediabetes with healthy subjects or subjects with diabetes is limited. The study aimed to determine corneal nerve fiber density and morphology and dendritic cell density between healthy subjects and those with prediabetes or type 2 diabetes.MethodsFifty-two subjects (aged 30–70 years) were recruited. Blood samples and body metrics were taken. Subjects were grouped as: healthy controls (hemoglobin A1c [HbA1c] < 5.7%), prediabetes (5.7–6.4%), and type 2 diabetes (> 6.4% or physician diagnosis). Central corneal subbasal nerve plexus was imaged using in vivo confocal microscopy. Corneal nerve fiber density and morphology, including interconnections and tortuosity, and dendritic cell density were assessed. Kruskal-Wallis tests were carried out to compare differences in the examined variables between groups. Spearman correlations were carried out to examine the associations between body metrics with HbA1c and corneal findings.ResultsSeventeen healthy controls, 20 subjects with prediabetes, and 15 subjects with type 2 diabetes completed this study. Central corneal nerve fiber density was significantly lower in type 2 diabetes compared to prediabetes (P = 0.045) and healthy controls (P = 0.001). No differences were found in central corneal nerve fiber interconnections, tortuosity, or dendritic cell density between groups. There was a significant association between HbA1c and corneal nerve fiber density (rho = −0.45, P = 0.001) and body mass index (BMI; rho = −0.30, P = 0.04).ConclusionsIncreased HbA1c values are associated with decreased corneal nerve fiber density across the spectrum of type 2 diabetes.

Corneal epithelial dendritic cells in patients with multiple sclerosis: An in vivo confocal microscopy study

PurposeTo evaluate density and morphology of corneal epithelial dendritic cells (DCs) in patients with multiple sclerosis (MS) using in vivo confocal microscopy (IVCM). MethodsThis was a single-center cross-sectional comparative study. All MS patients were clinically scored using the Expanded Disability Status Scale (EDSS) score. Patients underwent ophthalmological examination and then cornea was analyzed by IVCM Heidelberg Retina Tomograph (HRT 3) in combination with Rostock Cornea Module and CCD camera. Five sectors (central, nasal, temporal, inferior, superior and central area) were analyzed in both patient eyes, then for each sector one image was selected and analyzed by using the manual cell counting system offered with the software and ImageJ program. DCs density (cell/mm2) and DCs size (µm2) were considered for the analyses. Difference between the two groups and correlation between DCs, MS type, EDSS score, optic neuritis and ongoing therapy were analyzed. ResultsWe enrolled 46 consecutive patients: 23 with MS (age 47.87 ± 7.22 years (mean ± standard deviation) and 21 healthy subjects (age 46.0 ± 12.6 years) from July 2017 to July 2018. MS patients showed a lower DCs density when compared with healthy subjects (p < 0.05). Moreover, we found a direct correlation (r:0.48, p < 0.05) between DCs density and ongoing disease-modifying therapy. ConclusionIVCM was able to show a difference in corneal DCs density between MS patients and healthy subjects, providing an insight to the underlying changes of the clinical manifestations of MS. Further studies are needed to provide evidence of possible clinical implications.

Density and Morphology of Corneal Epithelial Dendritic Cells are Different in Allergy

ABSTRACTPurpose: The role of corneal epithelial dendritic cells (CEDC), a subtype of antigen presenting cells, in ocular allergy remains largely unknown. This cross-sectional study evaluated the density and morphology of CEDC in participants diagnosed with systemic allergy, to increase our understanding of the role of CEDC in ocular inflammation associated with systemic allergy.Materials and methods: A convenience sample of 50 participants was categorised into allergic and non-allergic groups (31 allergic and 19 non-allergic) based on the results of skin prick test (SPT). Ocular allergy symptoms, clinical ocular surface signs and serum IgE were assessed. In vivo confocal microscopy was performed on the right eye only. The number of CEDC in a 1mm2 region at both the central and mid-peripheral cornea was manually counted. CEDC morphology was graded on a 1 to 3 scale.Results: Ocular surface symptoms, signs (other than eyelid oedema), and serum IgE were significantly higher in the allergic (SPT+) group. CEDC density at the mid-peripheral cornea was significantly lower in the allergic group (p = .003). CEDC morphology grades were significantly higher in allergic participants in the central cornea (p = .02), with the highest grade morphology observed only in allergic participants. No associations were evident between CEDC density or morphology and ocular signs, symptoms or serum IgE.Conclusions: The study showed reduced CEDC density and cells with longer dendrites in allergic participants. The more mature CEDC morphology in the allergic group is suggestive of an inflammatory or immune response.

Corneal epithelial dendritic cell density in the healthy human cornea: A meta-analysis of in-vivo confocal microscopy data

PurposeNumerous studies have reported a wide range of corneal epithelial dendritic cells (CEDC) density using in-vivo confocal microscopy in healthy participants. It is necessary to establish normal CEDC values for healthy corneas to enable differentiation from pathological corneas. This meta-analysis aimed to establish CEDC density and distribution and examine their relationship with age and sex. MethodsA systematic review of the literature of studies using the Heidelberg Retinal Tomograph with Rostock Corneal Module and reporting CEDC density in healthy subjects up to December 2018 was conducted via Medline, Google Scholar, Scopus, PubMed, Embase and Cochrane library. A random effect modeling approach was used to obtain the results of meta-analysis and meta-regression was conducted to estimate the effect of age and sex. Results38 studies reported central and 9 reported peripheral inferior CEDC density of 1203 participants (mean age 46.0 ± 12.2, range 18–81 years). CEDC density in the central and peripheral inferior cornea was 26.4 ± 13.6 cells/mm2 (95% CI:22.5–26.8) and 74.9 ± 22.7 cells/mm2 (95%CI:59.8–90.0), respectively. No effect of age was found on central CEDC density (p = 0.63); whereas peripheral CEDC density decreased with increasing age (p = 0.02). CEDC density was not influenced by sex at either location (p > 0.48). ConclusionThis study established that the density at the peripheral inferior cornea is three-fold higher than at the central cornea. Peripheral but not central CEDC density decreased with increasing age. There are limited studies in youth (<18 years), precluding a more detailed analysis. Sex does not appear to be a significant factor in CEDC density.

Effect of age and contact lens wear on corneal epithelial dendritic cell distribution, density, and morphology

PurposeContact lens wearers aged 15–25 years are at higher risk of corneal inflammation, yet little is known about corneal inflammatory state in this group. Previous investigations show density of corneal epithelial dendritic cells (CEDC) may increase with contact lens wear. However, it is not known how corneal distribution or morphology of CEDC alters with lens wear or whether these markers are affected by age. This study characterised CEDC in adolescent and young adult contact lens wearers to determine effects of contact lens wear and age on CEDC density, distribution and morphology. MethodForty participants (20 contact lens wearers, 20 healthy non-wearers; age 16–36 years; 16M:24F) completed this pilot study. Corneal images were captured using in vivo confocal microscopy (HRTII, Rostock). CEDC were manually counted in a 1 mm2 area of the central and mid-peripheral cornea, and ratio of central to midperipheral density was calculated. CEDC morphology and dendrite length were graded. Differences between groups and between regions, and associations with age were examined. Significance was determined at P < 0.05. ResultsA lower ratio of central to mid-peripheral CEDC density was found with younger age (ρ = 0.42, P = 0.01). CEDC morphology was not associated with age or contact lens wear. CEDC density in the mid-peripheral cornea was higher in soft lens wearers than non-wearers (P = 0.04), but central density did not differ. CEDC density and morphology were not significantly different between centre (median density 11 cells/mm2, range 0–120) and mid-periphery (10 cells/mm2, 0–58). ConclusionDensity, distribution and morphology of CEDC do not differ in established contact lens wearers. A relatively lower density of CEDC in the central cornea of younger patients may allude to a more naive immune status in this group and warrants further study. Decreased central CEDC density identified in orthokeratology lens wear requires confirmation in a larger group.

Loss of Neurokinin-1 Receptor Alters Ocular Surface Homeostasis and Promotes an Early Development of Herpes Stromal Keratitis.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-γ-secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R-/- mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.

In Vivo Confocal Microscopy in Dry Eye Disease Associated With Chronic Graft-Versus-Host Disease.

To compare densities of corneal epithelial dendritic cells (DCs), corneal subbasal nerves, and conjunctival epithelial immune cells (EICs) between patients with dry eye disease (DED) with and without graft-versus-host disease (GVHD) using in vivo confocal microscopy (IVCM). This study included 54 patients who had moderate to severe DED either associated with (n = 33) or without (n = 21) chronic GVHD. In addition to evaluating clinical parameters of DED, images obtained by laser-scanning IVCM of the central cornea and superior tarsal conjunctiva were analyzed to measure densities of corneal epithelial DCs, corneal subbasal nerves, and conjunctival EICs. Although there were no significant differences between GVHD and non-GVHD groups in symptom scores, the GVHD group had significantly worse corneal fluorescein staining, tear break-up time, and Schirmer's scores than the non-GVHD group. Corneal epithelial DC density, corneal subbasal nerve density, and conjunctival EIC density were 148 ± 135 cells/mm2, 16.3 ± 6.1 mm/mm2, and 670 ± 267 cells/mm2, respectively, in the GVHD group; and 122 ± 99 cells/mm2, 18.3 ± 5.1 mm/mm2, and 572 ± 271 cells/mm2, respectively, in the non-GVHD group. After adjusting for clinical parameters, including the DED severity, none of the IVCM parameters was significantly different between the GVHD versus non-GVHD groups (P = 0.82, P = 0.21, and P = 0.60, respectively). In GVHD-associated DED, cellular changes in the cornea and conjunctiva observed by IVCM were similar to those seen in patients who have non-GVHD dry eye with the same level of disease severity. Therefore, corneal and conjunctival IVCM findings in GVHD-associated DED are possibly reflective of the local disease (DED) severity rather than the underlying systemic disease process.