Corneal Immune Cells Research Articles

Corneal inflammatory cell infiltration predicts disease activity in chronic inflammatory demyelinating polyneuropathy

The assessment of disease activity is fundamental in the management of chronic inflammatory demyelinating polyneuropathy (CIDP). Previous studies with small patient numbers found an increase of corneal immune cell infiltrates as a potential marker of inflammation in patients with CIDP. However, its clinical relevance remained unclear. The present study aimed to determine whether the amount of corneal inflammatory cells (CIC) measured by corneal confocal microscopy (CCM) detects disease activity in CIDP. CIC were measured in 142 CCM-investigations of 97 CIDP-patients. Data on clinical disease activity, disability (INCAT-ODSS) and need for therapy escalation at the timepoint of CCM, 3 and 6 months later were analyzed depending CIC-count. Pathological spontaneous activity during electromyography was examined as another possible biomarker for disease activity in comparison to CIC-count. An increased CIC-count at baseline was found in patients with clinical disease activity and disability progression in the following 3–6 months. An increase to more than 25 CIC/mm2 had a sensitivity of 0.73 and a specificity of 0.71 to detect clinical disease activity and a sensitivity of 0.77 and a specificity of 0.64 to detect disability progression (increasing INCAT-ODSS) in the following 6 months. An increase to more than 50 CIC/mm2 had a sensitivity of about 0.51 and a specificity of 0.91 to detect clinical disease activity and a sensitivity of 0.53 and a specificity of 0.80 to detect disability progression. CIC count is a non-invasive biomarker for the detection of disease activity in the following 6 months in CIDP.

Corneal immune cell morphometry as an indicator of local and systemic pathology: A review.

The corneal epithelium contains a population of resident immune cells commonly referred to as dendritic cells (DCs), or Langerhans cells. A unique advantage of the transparent cornea being situated at the surface of the eye is that these cells can be readily visualised using in vivo confocal microscopy. Over the past decade, interest in the involvement of corneal DCs in a range of ocular and systemic diseases has surged. For most studies, the number of corneal DCs has been the main outcome of interest. However, more recently attention has shifted towards understanding how DC morphology may provide insights into the inflammatory status of the cornea, and in some cases, the health of the peripheral nervous system. In this review, we provide examples of recent methodologies that have been used to classify and measure corneal DC morphology and discuss how this relates to local and systemic inflammatory conditions in humans and rodents.

Corneal Immune Cells Are Increased in Patients With Multiple Sclerosis.

PurposeCorneal confocal microscopy (CCM) is an ophthalmic imaging technique that has been used to identify increased corneal immune cells in patients with immune-mediated peripheral neuropathy. Given that multiple sclerosis has an immune-mediated etiology, we have compared corneal immune cell (IC) density and near-nerve distance in different subtypes of patients with multiple sclerosis (MS) to controls.MethodsThis is a blinded, cross-sectional study conducted at a tertiary hospital. Patients with clinically isolated syndrome (CIS) (n = 9), relapsing-remitting multiple sclerosis (RRMS) (n = 43), secondary progressive multiple sclerosis (SPMS) (n = 22), and control subjects (n = 20) underwent CCM. The total, mature, and immature corneal IC density and their nearest nerve distance were quantified.ResultsThe total IC density was higher in patients with MS (P = 0.02), RRMS (P = 0.01), and SPMS (P = 0.04) but not CIS (P = 0.99) compared to controls. Immature IC density was higher in patients with MS (P = 0.03) and RRMS (P = 0.02) but not SPMS (P = 0.10) or CIS (P = 0.99) compared to controls. Mature IC density (P = 0.15) did not differ between patients with MS and controls. The immature IC near-nerve distance was significantly greater in patients with MS (P = 0.001), RRMS (P = 0.007), and SPMS (P = 0.002) compared to controls. Immature IC density correlated with the Symbol Digit Modalities Test (r = –0.281, P = 0.02) and near-nerve distance correlated with the Expanded Disability Status Scale (r = 0.289, P = 0.005).ConclusionsIn vivo CCM demonstrates an increase in immature IC density and the near-nerve distance in patients with MS. These observations merit further studies to assess the utility of CCM in assessing neuroimmune alterations in MS.Translational RelevanceMultiple sclerosis is an immune-mediated neurodegenerative disease. Dendritic cells mediate communication between the innate and adaptive immune systems. We have used in vivo CCM to show increased corneal ICs and suggest it may act as an imaging biomarker for disease status in patients with MS.

The effect of high-fat diet-induced metabolic disturbance on corneal neuroimmune features

PurposeThe highly innervated cornea is susceptible to nerve loss secondary to systemic diseases such as diabetes and metabolic disturbances caused by high-fat diet. In this study, we characterize the effect of high-fat diet on the mouse corneal neuroimmune phenotype, including changes to corneal nerve density and resident immune cells, alongside the clinical assessment of corneal thickness and endothelial cell density. MethodsMale C57Bl6/J mice, aged 10 weeks, were fed a high-fat diet (60 kcal% fat, 5.2 kcal/g) or control diet (10 kcal%, 3.8 kcal/g) for 16 weeks. At the study endpoint, metabolic parameters (HbA1c, weight, fasting glucose, body fat) were measured to confirm metabolic disturbance. Clinical imaging of the anterior segment was performed using optical coherence tomography to measure the corneal epithelial and stromal thickness. Corneal sensory nerves were visualized using flatmount immunostaining and confocal microscopy. The topographical distribution and density of sensory nerves (BIII-tubulin+), intraepithelial CD45+ and MHC- II+ cells, stromal macrophages (IBA1+CD206+) and endothelial cells (ZO-1+) were analysed using FIJI. ResultsHigh-fat diet mice had significantly higher blood HbA1c, higher body weight, a higher percentage of body fat and elevated fasting glucose compared to the control diet mice. Corneal epithelial and stromal thickness was similar in both groups. The sum length of the basal nerve plexus was lower in the central and peripheral cornea of mice fed a high-fat diet. In contrast, the sum length of superficial nerve terminals was similar between groups. Epithelial immune cell density was two-fold higher in the central corneas of high-fat diet mice compared to control diet mice. IBA1+CD206+ macrophage density was similar in the anterior stroma of both groups but was significantly higher in the posterior stroma of the peripheral cornea in the high-fat diet mice compared to controls. The percentage of nerve-associated MHC-II+ cells in the epithelium and stroma was higher in HFD mice compared to controls. Endothelial cell density was similar in the corneas of high-fat diet mice compared to controls. ConclusionTogether with corneal neuropathy, corneal immune cells in mice fed a high-fat diet were differentially affected depending on their topographical distribution and location within cornea, and appeared in closer proximity to epithelial and stromal nerves, suggesting a local neuroimmune disruption induced by systemic metabolic disturbance.

Correlation of corneal immune cell changes with clinical severity in dry eye disease: An in vivo confocal microscopy study

PurposeTo evaluate corneal immune dendritiform cell (DC) changes in dry eye disease (DED) using in vivo confocal microscopy (IVCM) and to correlate IVCM parameters with clinical severity. MethodsThis was a retrospective, cross-sectional study including 300 eyes of 150 DED patients and 49 eyes of 49 age-matched controls. Severity of DED was based on the Dry Eye Workshop (DEWS) classification. IVCM images of subbasal layer of the central cornea were analyzed for DC density and morphology (including number of dendrites per DC, DC size and DC field). ResultsDC density was significantly higher in DED compared to controls (93.4 ± 6.3 vs. 25.9 ± 3.9 cells/mm2; P < 0.001). Morphologically, number of dendrites, DC size and field were significantly larger in DED (3.3 ± 0.1, 106.9 ± 4.7 μm2, 403.8 ± 20.1 μm2 than controls (2.3 ± 0.1, 62.5 ± 5.7 μm2, 241.4 ± 24.4 μm2, P < 0.001). Significantly higher DC density compared to controls was observed as early as Level 1 DED severity (87 ± 10 cells/mm2, p < 0.001. Significant morphological changes in DC were detected for Levels 2 to 4 (p=<0.001, and p =< 0.05) for dendrites and DC field, respectively. Similarly, DC size showed significant increase at DED level 3–4. (p < 0.05). Linear regression analysis showed that both conjunctival and corneal staining were independently associated with DC density, while corneal staining was independently associated with DC morphology. ConclusionDC density and morphology correlated with clinical severity of DED. While, DC density is increased in mild DED, morphological changes are seen only in severe cases. IVCM may be a powerful tool to detect early immune changes and may complement clinical examination in DED.

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

BackgroundTauopathy 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.MethodsCorneas 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.ResultsCompared 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.ConclusionsCorneal 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.

A Review of Imaging Biomarkers of the Ocular Surface.

A biomarker is a "characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions." Recently, calls for biomarkers for ocular surface diseases have increased, and advancements in imaging technologies have aided in allowing imaging biomarkers to serve as a potential solution for this need. This review focuses on the state of imaging biomarkers for ocular surface diseases, specifically non-invasive tear break-up time (NIBUT), tear meniscus measurement and corneal epithelial thickness with anterior segment optical coherence tomography (OCT), meibomian gland morphology with infrared meibography and in vivo confocal microscopy (IVCM), ocular redness with grading scales, and cellular corneal immune cells and nerve assessment by IVCM. Extensive literature review was performed for analytical and clinical validation that currently exists for potential imaging biomarkers. Our summary suggests that the reported analytical and clinical validation state for potential imaging biomarkers is broad, with some having good to excellent intra- and intergrader agreement to date. Examples of these include NIBUT for dry eye disease, ocular redness grading scales, and detection of corneal immune cells by IVCM for grading and monitoring inflammation. Further examples are nerve assessment by IVCM for monitoring severity of diabetes mellitus and neurotrophic keratitis, and corneal epithelial thickness assessment with anterior segment OCT for the diagnosis of early keratoconus. However, additional analytical validation for these biomarkers is required before clinical application as a biomarker.

FV 18 Corneal immune cell infiltration in corneal confocal microscopy confirm as biomarker for disease activity in autoimmune inflammatory neuropathies

FV 18 Corneal immune cell infiltration in corneal confocal microscopy confirm as biomarker for disease activity in autoimmune inflammatory neuropathies

In Vivo Confocal Microscopy Demonstrates Increased Immune Cell Densities in Corneal Graft Rejection Correlating With Signs and Symptoms

Diagnosis of graft rejection is based on patient symptoms and on clinical signs detected by slit-lamp biomicroscopy. This study investigated whether laser invivo confocal microscopy (IVCM) can aid in the diagnosis of corneal graft rejection by detecting cellular corneal changes that take place after transplantation. Prospective case-control study. Thirty-eight eyes of 38 patients with penetrating keratoplasty (15 eyes with corneal graft rejection, 23 eyes without rejection) and 9 age-matched normal controls. Laser IVCM was performed in the corneal grafts centrally. The density of immune cells (IC) was assessed for epithelial, sub-epithelial, stromal, and endothelial layers by 2 masked observers. IC density was compared among different groups and correlated to clinical signs and symptoms of corneal graft rejection. Outcome measurement was the IC density in the corneal layers and its associations with the presence of clinical signs and symptoms of corneal graft rejection. The IC density was significantly different between rejected and non-rejected grafts (P= 0.004) and different from that of normal controls (P= 0.001). Among corneal layers, IC density was significantly higher in rejected grafts than in non-rejected grafts in only the sub-basal (611.54 ± 573.74 vs. 340.61 ± 268.60 cells/mm2, respectively; P= 0.049) and endothelial layers (250.62 ± 267.13 vs. 103.47 ± 81.91 cells/mm2, respectively; P= 0.001). Patients with decreased best corrected visual acuity, Khodadoust line, and anterior chamber cells demonstrated a significant increase in total IC density (P < 0.05), whereas patients with symptoms of irritation, light sensitivity, and pain revealed a specific increase in IC density in the sub-basal layer (P < 0.05). Patients with ocular pain had higher IC density in the epithelial layer than those without pain (P= 0.03). Patients with corneal graft rejection demonstrate a significant increase in corneal immune cells, particularly, in the sub-basal and endothelial layers compared to patients with non-rejected grafts and controls. Although symptoms associated with endothelial rejection demonstrate a general increase in IC, pain, irritation, and light sensitivity are associated with increased IC in the sub-basal layer. Assessment of patients with corneal graft rejection by IVCM may serve as an adjunctive tool in the diagnosis and management of corneal graft rejection.

In vivo confocal microscopy detects bilateral changes of corneal immune cells and nerves in unilateral herpes zoster ophthalmicus

PurposeTo analyze bilateral corneal immune cell and nerve alterations in patients with unilateral herpes zoster ophthalmicus (HZO) by laser in vivo confocal microscopy (IVCM) and their correlation with corneal sensation and clinical findings. Materials and methodsThis is a prospective, cross-sectional, controlled, single-center study. Twenty-four eyes of 24 HZO patients and their contralateral clinically unaffected eyes and normal controls (n = 24) were included. Laser IVCM (Heidelberg Retina Tomograph/Rostock Cornea Module), corneal esthesiometry (Cochet-Bonnet) were performed. Changes in corneal dendritiform cell (DC) density and morphology, number and length of subbasal nerve fibers and their correlation to corneal sensation, pain, lesion location, disease duration, and number of episodes were analyzed. ResultsHZO-affected and contralateral eyes showed a significant increase in DC influx of the central cornea as compared to controls (147.4 ± 33.9, 120.1 ± 21.2, and 23.0 ± 3.6 cells/mm2; p < 0.0001). In HZO eyes DCs were larger in area (319.4 ± 59.8 μm2; p < 0.001) and number of dendrites (3.5 ± 0.4 n/cell; p = 0.01) as compared to controls (52.2 ± 11.7, and 2.3 ± 0.5). DC density and size showed moderate negative correlation with total nerve length (R = −0.43 and R = −0.57, respectively; all p < 0.001). A higher frequency of nerve beading and activated DCs close to nerve fibers were detected specifically in pain patients. ConclusionsChronic unilateral HZO causes significant bilateral increase in corneal DC density and decrease of the corneal subbasal nerves as compared to controls. Negative correlation was observed for DC density and size to nerve parameters, suggesting interplay between the immune and nervous systems. Patients with chronic pain also showed increased nerve beading and activated DCs.

Bilateral Alterations in Corneal Nerves, Dendritic Cells, and Tear Cytokine Levels in Ocular Surface Disease.

This review summarizes the recent literature regarding corneal imaging in human subjects using in vivo confocal microscopy. It also covers the recent literature on corneal immune cells, nerves, and tear cytokine levels in ocular surface diseases as well as corneal immune privilege. The significance of interactions between corneal immune cells and nerves in health, neurotrophic keratopathy, and infectious keratitis is discussed. Furthermore, bilateral alterations of immune cells and nerves in clinically unilateral corneal diseases and the link to changes of tear cytokines or neuropeptide levels in contralateral eyes are described. Recent studies reported increased density and morphologic changes of corneal dendritic cells in ocular surface disease that correlated with a decrease in subbasal nerve and corneal nerve density, suggesting potential interactions between the immune and nervous systems in the cornea. Although the relevance of tear cytokines is poorly understood, tear cytokines might have an important role in the pathogenesis of ocular surface diseases. In humans and experimental animal models, alterations in immune cells, cytokines, and immunomodulatory neuropeptide levels in contralateral eyes might mediate the incidence of bilateral infectious keratitis and loss of immune privilege of the cornea in bilateral corneal transplantation or neurotrophic keratopathy cases. The discovery of bilateral alterations of immune cells and nerves in ocular surface diseases is considered the missing link between the immune and nervous systems in the cornea, and demonstrates how studies of animal models and humans aid our understanding of human corneal disease phenomena.

Translational Immunoimaging and Neuroimaging Demonstrate Corneal Neuroimmune Crosstalk.

Corneal immunoimaging and neuroimaging approaches facilitate in vivo analyses of the cornea, including high-resolution imaging of corneal immune cells and nerves. This approach facilitates the analyses of underlying immune and nerve alterations not detected by clinical slit-lamp examination alone. In this review, we describe recent work performed in our translational ocular immunology center with a focus on "bench-to-bedside" and "bedside-to-bench" research. The ability to visualize dendritiform immune cells (DCs) in patients with laser in vivo confocal microscopy (IVCM), recently discovered in the central murine cornea, has allowed us to demonstrate their utility as a potential surrogate biomarker for inflammatory ocular surface diseases. This biomarker for inflammation allows the measurement of therapeutic efficacy of anti-inflammatory drugs and its utility as an endpoint in clinical trials with high interobserver agreement. IVCM image analyses from our studies has demonstrated a significant increase in DC density and size in ocular disease, a positive correlation between DC density and clinical signs and symptoms of disease and pro-inflammatory tear cytokines, and a strong negative correlation between DC density and subbasal nerve density. In conjunction with preclinical research investigating the inflammatory state in a partial or fully denervated cornea, our results indicated that corneal nerves are directly involved in the regulation of homeostasis and immune privilege in the cornea.

EP 41. Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathies

Objective There is an unmet need for better diagnostic tools to further delineate clinical subsets of heterogeneous chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN) to facilitate treatment decisions. Corneal confocal microscopy (CCM) is a non-invasive and reproducible nerve imaging technique. This study evaluates the potential of CCM as a diagnostic surrogate in CIDP and MMN. Methods In a cross-sectional prospective approach 182 patients and healthy controls were studied using CCM to quantify corneal nerve damage and immune cell infiltration. Results Patients with CIDP and MMN had a reduction in corneal nerve fiber (CNF) measures and an increase in corneal immune cell infiltrates. In CIDP, CNF parameters decreased with increasing duration of disease. The number of dendritic cells in proximity to corneal nerve fibers was increased in patients with early disease and correlated with the degree of motor affection. A further reduction in CNF parameters and an increase in non-dendritic cells was observed in patients with painful neuropathy. In CIDP patients with anti-neuronal antibodies the number of non-dendritic cells was increased. Interpretation Our findings suggest that corneal nerve fiber loss may reflect severity of neuropathy and quantification of distinct cells around the CNF plexus may help in stratifying CIDP subtypes, clinical course and disease activity. However, further longitudinal studies are required before CCM can be considered as a valid surrogate end point for patients with CIDP and MMN.

Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathy.

There is an unmet need for better diagnostic tools to further delineate clinical subsets of heterogeneous chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN) to facilitate treatment decisions. Corneal confocal microscopy (CCM) is a noninvasive and reproducible nerve imaging technique. This study evaluates the potential of CCM as a diagnostic surrogate in CIDP and MMN. In a cross-sectional prospective approach, 182 patients and healthy controls were studied using CCM to quantify corneal nerve damage and immune cell infiltration. Patients with CIDP and MMN had a reduction in corneal nerve fiber (CNF) measures and an increase in corneal immune cell infiltrates. In CIDP, CNF parameters decreased with increasing duration of disease. The number of dendritic cells in proximity to CNFs was increased in patients with early disease and correlated with the degree of motor affection. A further reduction in CNF parameters and an increase in nondendritic cells were observed in patients with painful neuropathy. In CIDP patients with antineuronal antibodies the number of nondendritic cells was increased. Our findings suggest that CNF loss may reflect severity of neuropathy and quantification of distinct cells around the CNF plexus may help in stratifying CIDP subtypes, clinical course, and disease activity. However, further longitudinal studies are required before CCM can be considered as a valid surrogate endpoint for patients with CIDP and MMN.

Herpesvirus entry mediator on radiation-resistant cell lineages promotes ocular herpes simplex virus 1 pathogenesis in an entry-independent manner.

ABSTRACTOcular herpes simplex virus 1 (HSV-1) infection leads to a potentially blinding immunoinflammatory syndrome, herpes stromal keratitis (HSK). Herpesvirus entry mediator (HVEM), a widely expressed tumor necrosis factor (TNF) receptor superfamily member with diverse roles in immune signaling, facilitates viral entry through interactions with viral glycoprotein D (gD) and is important for HSV-1 pathogenesis. We subjected mice to corneal infection with an HSV-1 mutant in which HVEM-mediated entry was specifically abolished and found that the HVEM-entry mutant produced clinical disease comparable to that produced by the control virus. HVEM-mediated induction of corneal cytokines, which correlated with an HVEM-dependent increase in levels of corneal immune cell infiltrates, was also gD independent. Given the complexity of HVEM immune signaling, we used hematopoietic chimeric mice to determine which HVEM-expressing cells mediate HSV-1 pathogenesis in the eye. Regardless of whether the donor was a wild-type (WT) or HVEM knockout (KO) strain, HVEM KO recipients were protected from ocular HSV-1, suggesting that HVEM on radiation-resistant cell types, likely resident cells of the cornea, confers wild-type-like susceptibility to disease. Together, these data indicate that HVEM contributes to ocular pathogenesis independently of entry and point to an immunomodulatory role for this protein specifically on radiation-resistant cells.

Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

BackgroundThe role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.Methodology/Principal FindingsWe now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.ConclusionsTo our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.