Tear Cytokines Research Articles

Ocular tear fluid biomarkers collected by contact lenses

PurposeTo collect tear fluid biomarkers from contact lenses (CLs) and determine the impact of CL wear duration. MethodsRabbits were fitted with commercial etafilcon A CLs, which were collected after 1 min, 4 and 8 h (n = 4/time point). Tear fluid proteins and cytokines were extracted from the CLs and quantified. An exploratory comparison was performed between CLs and Schirmer Strips (SS) for a 1 min duration. ResultsThe concentration of MUC5AC was significantly higher after 4 h of CL wear. The expression of all investigated cytokines (IL-1α, IL-1β, IL-8, IL-17A, IL-21, Leptin, MIP-1β, MMP-9, NCAM-1, and TNF-α) was detectable after 1 min of CL wear, and over time, all showed significant variations throughout the 8-h CL wear period. Notably, IL-1α significantly increased by 8 h of CL wear, while MMP-9 decreased. Albumin and lysozyme did not show significant variations with CL wear. Differences between CLs and SS after 1 min were statistically significant for albumin, Leptin, TNF-α, IL-1α, IL-1β, and IL-8. ConclusionsThe duration of CL wear significantly affects the collection of some tear fluid biomarkers. Albumin, MUC5AC, and cytokines may have individual and synergistic diagnostic or prognostic potential. CLs and SS were similar for lysozyme and MUC5AC but differed in the collection of albumin and some cytokines. CLs are a viable tear fluid collection method for biomarker analyses and can be immediately added as a routine clinical test by being FDA-approved medical devices.

Longitudinal Analysis of Tear Cytokine Ratios in Association with Dry Eye Signs and Symptoms in the Dry Eye Assessment and Management (DREAM) Study

Purpose To extend cross-sectional data on cytokine ratios (CRs) in dry eye disease (DED) signs by investigating longitudinal change in pro- to anti-inflammatory CRs and associations with change in DED signs and symptoms. Methods Secondary analysis of fifty-four subjects [mean age 57.3 (SD 13.2) years, 85.2% female; 68.5% white] with ≥ 4 uL pooled tear volumes at months 0, 6, and 12. Pro-inflammatory (IL-1b, IL-6, IL-8, IL-17A, IFN-g, and TNF-a) to anti-inflammatory (IL-6, IL-10) cytokine ratios (CR) were calculated. DED signs (corneal and conjunctival staining scores, tear break-up time, Schirmer test, Meibomian gland plugging, tear osmolarity, composite sign severity score) and symptoms [Ocular Surface Disease Index (OSDI)] were measured. Changes over time in DED signs, symptoms, and CRs were evaluated using longitudinal models. Correlations between changes in CR and changes in DED signs and symptoms were evaluated using Spearman correlation coefficients (rho). Results DED signs which improved over time (p < 0.001) included corneal and conjunctival staining score, tear break-up time, tear osmolarity, and composite sign severity score. Using IL-10 as anti-inflammatory, changes in corneal and conjunctival staining and composite severity score significantly correlated with changes in pro- to anti-inflammatory CRs from month 0 to 6 (|rho|: 0.29-0.45, p: 0.003-0.04) but not between month 0 to 12 (|rho|: 0.01 to 0.24, all p > 0.08). DED symptoms decreased across one year (p < = 0.001) for all OSDI measures; these changes did not correlate with changes in CRs (|rho|: 0.00 to 0.29, all p > 0.04). Conclusions Improvement in some DED signs across one year correlated weakly with decreases in pro- to anti-inflammatory CRs, in alignment with the understanding of DED as inflammatory. CRs may provide greater insight than absolute tear cytokine concentrations as possible DED biomarkers. Additional studies that provide standardized clinical information and tear collection and analysis are needed to validate CRs in DED.

Relationship of Inflammatory Mediators (Interleukin and Cortisol Concentrations) with Corneal Epithelial Quantifiable Metrics

PurposeTo investigate the relationship of inflammatory biomarkers with corneal epithelial quantifiable metrics in patients with keratoconus and in healthy eyes. DesignProspective observational comparative study. ParticipantsThis study included 100 eyes of 100 patients, 48 eyes of 48 patients with KC, and 52 healthy eyes of 52 healthy controls. MethodsThe concentrations of tear cytokines were investigated in both groups: interleukin (IL) 1B, IL6, IL8, IL10, IL12p70 and TNFα were obtained by capillary flow and measured using flow cytometer. Cortisol concentrations were determined in both groups from the most proximal hair segment as an index of cumulative secretion and measured by liquid chromatography mass spectrometry. Epithelial variables were obtained with optical coherence tomography (OCT). Pearson correlation (r) was used to measure linear dependence between two different variables. Main Outcome MeasuresInvestigating the existence, strength, and significance of any correlation between inflammatory biomarkers (IL1B, IL6, IL8, IL10, IL12p70, TNFα and hair cortisol concentration) and OCT corneal epithelial quantifiable variables such as minimum and maximum epithelial thickness of the map, difference between the minimum and maximum (Epithelial Min-Max) and standard deviation of the epithelial thickness of the map (Epithelial Std Dev) and average epithelial thickness of the superior and inferior regions of the map. ResultsEyes with keratoconus presented statistically significantly higher levels of IL1b (p=0.02), IL6 (p <0.0001), IL8 (p <0.0001), and TNFα (p <0.0001) and hair cortisol concentration (p=0.01) compared to healthy controls.There was a significant correlation between IL6 and measurement Epithelium Min-Max [Pearson= -0.59 (-0.69, -0.47); p<0.0001] and Epithelial standard deviation (Std Dev) [Pearson= +0.56 (0.44, 0.67); p<0.0001]. There was a significant correlation between hair cortisol concentration and Epithelium Min-Max [Pearson=-0.27 (-0.42, -0.1); p<0.0001] and Epithelium Std Dev groups [Pearson= +0.2 (0.03, 0.36); p=0.021]. There was also a significant correlation between TNFα and Epithelial Max. [Pearson= -0.37 (-0.55, 0.17); p<0.0001]. We found no significant correlation between the concentration of IL1b, IL8, IL10 and IL2p70 with any epithelium parameters. ConclusionsThe higher concentration of inflammatory markers (IL6 and hair cortisol) in eyes with keratoconus present a significant correlation with OCT metrics identifying epithelial variability, such as Epithelial Min-Max and Std Dev. These findings demonstrate the role of chronic inflammation in eyes with keratoconus, and that these epithelial changes detectable with OCT are sensitive to this inflammatory process.

Differential Ophthalmological Profile in Patients with Coronary Artery Disease Coexisting with Type 2 Diabetes Mellitus: Elevated Tear Cytokine Concentrations.

Background/Objectives: Coronary artery disease (CAD) and type-2 diabetes mellitus (T2DM) are characterized by chronic low-grade inflammation. However, measuring cytokines typically involves invasive blood sampling, which can be problematic for CAD patients. This study aimed to assess ophthalmological parameters and tear cytokines in patients with CAD, comparing those with comorbid T2DM to those without to understand their inflammatory profiles. Methods: One hundred subjects with suspected chronic or acute CAD were initially included in this single-center cross-sectional study after clinical stabilization. Seventy-two patients with confirmed CAD were divided into two groups: 32 patients with T2DM and 40 patients without T2DM. A total of 144 eyes were examined, and tear fluid samples were collected to determine cytokine concentrations. Ophthalmological parameters and tear concentrations of cytokines were analyzed, controlling for age, sex, and other cardiovascular risk factors. Results: Patients with CAD and T2DM exhibited decreased ophthalmological parameters and increased cytokine concentrations in comparison to those without T2DM. Significant inverse correlations between ophthalmological parameters and cytokine concentrations were observed. Following adjustment, a full logistic regression model for distinguishing patients with CAD and comorbid T2DM included macular cube volume, mean macular thickness, interleukin (IL)-4, IL-5, IL-6, IL-8, IL-9, IL-13, granulocyte colony-stimulating factor (G-CSF), CCL3, CCL4, and CCL11/eotaxin-1, demonstrating excellent discriminatory power (Area Under the Curve = 0.95, 95% Confidence Interval = 0.91-0.99; p < 0.001). Subsequently, IL-5 (Odds Ratio = 1.68, 95% CI = 1.26-2.24; p < 0.001), G-CSF (OR = 1.06, 95% CI = 1.02-1.11; p < 0.01), and CCL11/eotaxin-1 (OR = 1.56, 95% CI = 1.19-2.05; p = 0.001) emerged as the most distinguishing variables in a reduced model (AUC = 0.89, 95% CI = 0.84-0.95; p < 0.001). Conclusions: Differences in ophthalmological variables, mainly in cytokine concentrations, suggest distinct pathophysiological mechanisms in patients with CAD based on the presence of T2DM. These findings demonstrate that the inflammatory profile can be readily detected through tear sample cytokines, proving valuable for establishing more accurate prognoses and monitoring in cardiometabolic disorders.

Tear cytokines as potential markers of inflammation in feline conjunctivitis.

Conjunctivitis is a prevalent feline ocular surface disorder, often accompanied by inflammation. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a crucial role in the pathogenesis of conjunctival inflammation. This study aimed to evaluate the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis and compare them with healthy controls, thereby enhancing our understanding of the inflammatory processes in feline conjunctivitis. Tear samples were collected from cats of various breeds diagnosed with conjunctivitis (n = 15) and healthy control cats (n = 5) using Schirmer strips. The levels of TNF-α and IL-6 were measured using the enzyme-linked immunosorbent assay (ELISA) method. Protein concentration were measured using Bradford assay and data were expressed as pg/mg protein of tear sample. Our results revealed a statistically significant increase in the levels of both TNF-α and IL-6 in cats with conjunctivitis compared to the control group (p < .0001). Positive correlation were observed between tear IL-6 (p < .001, r = 0.902) and TNF-α (p < .001, r = 0.919) with clinical grades of conjunctivitis. The results demonstrated a significant elevation in the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis, suggesting that these cytokines are involved in the inflammatory response of feline conjunctivitis. These findings could pave the way for new diagnostic and therapeutic approaches, focusing on cytokine modulation, to manage feline conjunctivitis more effectively.

Ocular involvement in Steven-Johnson syndrome/toxic epidermal necrolysis: recent insights into pathophysiology, biomarkers, and therapeutic strategies.

To review the pathophysiology, recent biomarkers related to the ocular aspects of Steven-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), and to highlight notable evidence published in recent years. Several studies reveal the relationship between tear cytokines and the pathological components in eyes of SJS/TEN patients. Specific clinical features and associated risk factors in the acute stage have shown significant correlations with chronic ocular sequelae. Recent treatment protocols, including early pulse systemic and topical steroids, as well as tumor necrosis factor-α inhibitors, have demonstrated positive effects on ocular outcomes. In addition to conventional surgical treatment, a new surgical technique, simple oral mucosal epithelial transplantation (SOMET), has been introduced as a simple ocular surface reconstruction for patient with SJS. Advancements in knowledge and management strategies have notably enhanced ocular outcomes for SJS/TEN eyes. A deeper understanding of the biomarker changes in these eyes could facilitate the development of future targeted treatment options.

Multimodal mucosal and systemic immune characterization of a non-human primate trachoma model highlights the critical role of local immunity during acute phase disease.

Trachoma is a leading cause of infection-related blindness worldwide. This disease is caused by recurrent Chlamydia trachomatis (Ct) infections of the conjunctiva and develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term: ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and severity of reinfections. The immune system plays a pivotal role in trachoma including exacerbation of the disease. Hence the immune system may also be key to developing a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. The conjunctiva of non-human primate (NHP, Cynomolgus monkeys-Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis indicated an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, followed by a late phase monocytic influx accompanied with a decrease in other immune cells, and tear fluid cytokines returning to initial levels. Our NHP model accurately reproduces the clinical signs of acute trachoma, allowing for an accurate assessment of the local immune responses in infected eyes. A progressive immune response occurred for weeks after exposure to Ct, which subsided into a persistent innate immune response. An understanding of these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies for disease prevention.

Clinical Characteristic and Tear Film Biomarkers After Myopic FS-LASIK: 1-Year Prospective Follow-up.

To assess the long-term (1-year) effect of myopic femtosecond laser-assisted in situ keratomileusis (FSLASIK) on clinical characteristics and tear film biomarkers. Eighty eyes from 80 patients who underwent FSLASIK were evaluated. Ocular surface symptoms and signs were evaluated using specific questionnaires and tests. The corneal nerves and dendritic cells were examined using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Tear inflammatory cytokines and neuropeptides were evaluated using Luminex immunoassay. These examinations were performed preoperatively and at 1, 3, 6, and 12 months postoperatively. Seventy-three participants completed all follow-up visits. Following FS-LASIK, ocular symptoms and signs (except Schirmer I test) worsened at 1 month but corneal and conjunctival stainings improved by 3 months. The numbers of dendritic cells and activated dendritic cells increased at the 3-month postoperative visit and recovered to preoperative levels by the 6-month visit. Ocular symptoms and corneal sensitivity recovered to preoperative levels at the 12-month visit. Tear break-up time and corneal nerve morphology were not recovered to preoperative status at the 12-month visit. Interleukin (IL)-1β, IL-17A, tumor necrosis factor-α, and substance P tear levels significantly increased at all postoperative visits compared to preoperative levels. Corneal staining scores positively correlated with tear IL-1β and IL-17A levels, whereas corneal nerve morphology positively correlated with corneal sensitivity and negatively correlated with substance P levels. Although most clinical variables improved at 12 months postoperatively, some tear inflammatory cytokines and substance P remain altered beyond 12 months, indicating that ocular homeostasis is not completely recovered. [J Refract Surg. 2024;40(8):e508-e519.].

Tear fluid cytokine analysis: a non-invasive approach for assessing retinopathy of prematurity severity.

To determine whether there is a significant association between inflammatory cytokines in the tear fluid and the severity of Retinopathy of Prematurity (ROP). Retrospective cohort study. The cytokine levels in tear fluids were determined in 34 eyes with ROP and 18 eyes without ROP. There were 15 eyes with severe ROP requiring treatment and 19 eyes with mild ROP not requiring treatment. For severe ROP eyes, tear fluids were collected before treatment. Significantly higher levels of CCL2 and vascular endothelial growth factor (VEGF) were detected in eyes with severe ROP compared to eyes with mild ROP and no ROP. When assessed for cytokine levels that discriminate each disease group, CCL2 showed a significant odds ratio of 1.76 for severity change (/quintile, P = 0.032, after adjusting for birth weight). Correlation analysis showed that birth weight correlated with IL-1α levels, and decreased weight gain increased IFN-γ levels. We next determined tear fluid cytokines which discriminate severe ROP using receiver operating characteristics' analysis. We found that combination of higher CCL2 levels, higher VEGF levels, and lower IFN-γ levels in the tear fluid had a stronger predictive value for severe ROP (area under curve, 0.85). The levels of CCL2, VEGF, and IFN-γ in tear fluid may serve as useful biomarkers for assessing the severity of ROP.

Tear Cytokines as Predictive Biomarkers of Success in Contact Lens Discomfort Management

ABSTRACT Purpose To analyze changes in tear levels of inflammatory mediators in symptomatic contact lens (CL) wearers after refitting with daily disposable CLs and to identify potential biomarkers of success in CL discomfort (CLD) management. Methods Symptomatic CL wearers (CLDEQ-8 ≥ 12) were refitted (V1) with daily disposable CLs (Delefilcon A). After one month (V2), participants were classified into the post-fitting non-symptomatic (CLDEQ <12) and symptomatic (CLDEQ ≥12) groups. At each visit, the participants were clinically evaluated, tears were collected, and 20 inflammatory mediators and substance P (SP) were measured using multiplex immunobead analysis and ELISA, respectively. The detection rates and concentrations were compared between visits and groups, and logistic regression models were performed. Results Forty-three subjects (32 women/11 men; mean age: 23.2 ± 4.9 years) were enrolled. The IL-1β and IL-9 detection rates were higher at V2 (p ≤ 0.044). The detection rates of IL-1β, IL-9, MIP-1α/CCL3, and MMP-9 at V1 (p ≤ 0.045) and IL-17A at V2 (p ≤ 0.014) were higher in the post-fitting symptomatic group. The tear IL-9 concentration was increased at V2 (p = 0.018). The tear concentrations of fractalkine/CX3CL1, IL-2, IL-6, IL-10, MCP-3/CCL7, MIP-1β, NGF, RANTES/CCL5, and TNF-α were higher in the post-fitting symptomatic group (p ≤ 0.044). Additionally, levels of fractalkine/CX3CL1, IL-2, IL-6, IL-10, RANTES/CCL5, and TNF-α at V1 were significantly associated with the post-fitting grouping (p ≤ 0.044). Conclusions Low tear concentrations of specific inflammatory mediators may be used as a predictive biomarker of success for refitting symptomatic CL wearers with daily disposable CLs. However, complementary treatments might be required for symptomatic CL wearers with higher levels of these inflammatory molecules.

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

Defining how the invivo immune status of peripheral tissues is shaped by the external environment has remained a technical challenge. We recently developed Functional invivo 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. Longitudinal, observational clinical study. 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]). 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. Difference in the density, morphology, 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 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. This study identifies that the invivo 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 invivo window to investigate season-dependent environmental influences on the human immune system. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Framework Nucleic Acids Combined with 3D Hybridization Chain Reaction Amplifiers for Monitoring Multiple Human Tear Cytokines.

Existing tear sensors are difficult to perform multiplexed assays due to the minute amounts of biomolecules in tears and the tiny volume of tears. Herein, the authors leverage DNA tetrahedral frameworks (DTFs) modified on the wireless portable electrodes to effectively capture 3D hybridization chain reaction (HCR) amplifiers for automatic and sensitive monitoring of multiple cytokines in human tears. The developed sensors allow the sensitive determination of various dry eye syndrome (DES)-associated cytokines in human tears with the limit of detection down to 0.1 pgmL-1, consuming as little as 3 mL of tear fluid. Double-blind testing of clinical DES samples using the developed sensor and commercial ELISA shows no significant difference between them. Compared with single-biomarker diagnosis, the diagnostic accuracy of this sensor based on multiple biomarkers has improved by ≈16%. The developed system offers the potential for tear sensors to enable personalized and accurate diagnosis of various ocular diseases.

Vitamin D and tear fluid cytokines in predicting outcomes in viral conjunctivitis - A new outlook.

To determine the association between systemic vitamin D (VD) and immunoglobulin E (IgE) levels with severity and ocular surface inflammatory profile in patients with epidemic keratoconjunctivitis (EKC). 210 eyes of 105 patients who were clinically diagnosed with EKC were included in the study. The levels of serum VD and serum IgE were measured. Schirmer's strip-based tear fluid (TF) was used to determine levels of IL-1β, IL-6, IL-10, IL-17A, TNFα, MMP9, sICAM1, and VEGF-A in a subset of patients. Levels of VD were significantly ( P < 0.05) lower and levels of IgE were significantly higher in patients with severe forms of conjunctivitis compared to those with nonsevere forms. Majority of the patients with severe forms of the disease exhibited VD deficiency and/or abnormally high IgE. A negative correlation (r = -0.682; P < 0.0001) was observed between VD and IgE levels. TF levels of IL-1β, IL-6, TNFα, and sICAM1 were significantly higher in eyes with severe forms of conjunctivitis compared to those with nonsevere forms and controls. These factors showed a positive correlation ( P < 0.05) with IgE levels and a negative correlation ( P < 0.05) with VD levels. Patients with severe forms of EKC exhibited VD deficiency and higher levels of IgE. Increased TF inflammatory factors demonstrated a disease causal relationship with VD and IgE. Hence, restoring the altered levels of VD and IgE to normal range would be pivotal in the prevention and management of severe conjunctivitis.

The level of cytokines in tears as a novel indicator of demyelinating diseases

ABSTRACT Introduction A novel research objective is to identify new molecules in more readily accessible biological fluids that could be used in the diagnosis of multiple sclerosis (MS) and other demyelinating disorders. Aim To compare the level of selected cytokines in tears between patients with MS or other demyelinating disorder and healthy controls. Material and methods 84 patients with diagnosed MS during remission or with other demyelinating disease of the CNS and 70 healthy controls were enrolled in the study. Tears were collected without any stimulation and stored till the day of assessment. The concentration of selected cytokines was measured by the Bio-Plex Pro Human cytokine screening panel 27 cytokines assay according to the manufacturer’s instructions. Statistical analysis was performed with Statistica 13. Results IL-1b level was significantly lower in the study group compared to the control group [3,6 vs 8.71, p < 0.001]. The same pattern was observed for IL-6 [3,1 vs 5.26, p = 0.027] and IL-10 [1,7 vs 10.92, p < 0.001] (Table 1). In the study group, IL-1RA (p = 0.015), IL-5 (p = 0.04), IL-9 (p = 0.014), and IL-15 (p = 0.037) showed significant correlations with age. In the total sample, IL-1Ra (p = 0.016) and IFN-g (p = 0.041) were significantly correlated with age, while in the control group, IL-8 (p = 0.09), MIP-1a (p = 0.009), and RANTES (p = 0.031) showed significant correlations. Conclusions Our results show that MS and other demyelination diseases lead to decrease in the overall level of cytokines in tears. Further research is needed to determine the role of tear fluid in the assessment of demyelinating disorders like MS.

Is Meibomian Gland Dysfunction One Disease? Heterogeneity Among Phenotypes.

The purpose of this study was to examine ocular surface symptoms, tear metrics, and tear cytokines by Meibomian gland dysfunction (MGD) features. Symptom questionnaires and an ocular surface evaluation were performed on 40 individuals with varied MGD signs (Meibomian gland [MG] plugging, eyelid vascularity, meibum quality, and MG dropout). Tear proteins were extracted off Schirmer strips and analyzed for 23 human inflammation-related proteins. Statistical analysis was performed to examine associations between dry eye metrics inflammatory proteins and MGD features. The study involved 40 South Florida veterans with a mean age of 61 ± 13 years; most individuals were male (95%), White (31%), and non-Hispanic (85%). MGD features differentially related to dry eye signs. Eyelid vascularity, meibum quality, and MG dropout, but not MG plugging, correlated with higher corneal staining and lower tear production. MGD features also differentially related to tear cytokines. Eyelid vascularity most closely related to inflammation with significant correlations for interferon-gamma-γ (r = 0.36, P = 0.02), interleukin-4 (IL-4) (r = 0.43, P = 0.006), IL-17A (r = 0.42, P = 0.007), matrix metalloproteinase-2 (r = 0.39, P = 0.01), C-X-C motif chemokine ligand 5 (Regulated upon Activation, Normal T-Cell Expressed and presumably Secreted [RANTES]) (r = 0.32, P = 0.04), and tumor necrosis factor α (r = 0.36, P = 0.02). The other 3 MGD signs were less related to inflammation. Multivariable models revealed IL-4 to be most closely related to eyelid vascularity (standardized β = 0.39, P < 0.0001). Eyelid vascularity was the MGD sign most closely related to inflammatory cytokines, suggesting that different MGD features may be driven by different pathophysiological mechanisms.

5 Janus Kinase 1/2 inhibition Effect on Cytokine Levels in Tears of Patients with Ocular Graft Versus Host Disease

OBJECTIVES/GOALS: The goal of this study is to identify if ocular graft versus host disease (oGVHD) patients treated with a systemic JAK inhibitor have a change in their tear cytokine profile (a possible bio-marker) and oGVHD score. oGHVD is a severe inflammatory dry eye disease and major cause of morbidity after a hematopoietic stem cell transplant. METHODS/STUDY POPULATION: Janus Kinase (JAK) is an upstream regulator of cytokine production. A JAK 1/2 inhibitor, Ruxolitinib, was recently FDA approved for the treatment of chronic GVHD. We propose that JAK inhibition results incytokine changes in tears and improvement of oGVHD. To study this, we will quantify tear cytokines in patients with oGVHD, with and without systemic JAK inhibition treatment. Patients with ‘definite’ oGVHD based on the international chronic oGVHD diagnostic criteria (ICOGVHD) whom we have collected tears will be grouped based on JAK inhibition treatment. Tear cytokines are analyzed using Iso spark Meteor bulk quantitative proteomic analysis. RESULTS/ANTICIPATED RESULTS: Seven patients were identified from our patient cohort who met inclusion criteria (oGVHD; tears collected while on Ruxolitinib), five patients were identified whom we have collected tears with oGVHD who have not taken ruxolitinib. The following 10 cytokines will be analyzed in the tears by the Iso spark Meteor bulk quantitative proteomic analysis: GM-CSF, IFN-g, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-a. The change in cytokine levels will be compared with the ICOGVHD score, corneal fluorescein staining, schirmers test (measurement of tear production), conjunctival injection score, ocular surface disease index score (validated symptomatic score of dry eye disease). DISCUSSION/SIGNIFICANCE: OGVHD is a major cause of morbidity for patients who undergo a hematopoietic stem cell transplant and is the result of a highly complex immune process including dysregulation of pro-inflammatory cytokines. It is critical to understand the effect of cytokine changes on the eyes to potentially identify a biomarker and possible treatment targets.

Tear cytokine levels are reduced in patients treated with intravitreal injections

PurposeTo investigate cytokine levels in the tear fluid of patients receiving serial intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF) for neovascular age-related macular degeneration (nAMD). MethodsConcentrations of six cytokines (IFN-γ, IL-1β, IL-6, IL-8, TNF and VEGF) in tears of patients receiving anti-VEGF in one eye were assayed using multiplex cytometric bead array. The fellow untreated eye served as control. Tear sampling was performed on a single occasion at a minimum of four weeks after IVI. Patients underwent a pre-IVI antisepsis protocol with povidone-iodine. ResultsTear fluid from thirty patients with a mean age of 78.8 years (range 58–90) was assayed. Subjects received a median of 43.5 (range 22–106) IVI in one eye. The median level of IFN-γ was 0.33 (interquartile range (IQR) 0.22–0.52) pg/mg of total protein in injected eyes versus 0.41 (IQR 0.21–1.05) pg/mg in fellow eyes (p = 0.017). For TNF, a median level of 0.12 (IQR 0.08–0.18) pg/mg of total protein was found in injected eyes versus 0.14 (IQR 0.07–0.33) pg/mg of total protein in fellow eyes (p = 0.019). There were no differences between injected and fellow eyes regarding the levels of IL-1β, IL-6, IL-8 and VEGF. ConclusionTear fluid in eyes receiving serial IVI with anti-VEGF and preoperative povidone-iodine antisepsis constitutes lower levels of the pro-inflammatory cytokines IFN-γ and TNF compared to fellow eyes. This provides biochemical support of previous findings of reduced signs of inflammation and healthier tear film parameters in patients treated with serial IVI.

Heritability of tear fluid cytokines in healthy twins

PurposeOcular surface disease is common and it is associated with elevated concentration levels of cytokines in tear fluid. Studies of the normal variation in tear fluid inflammatory markers are lacking. New knowledge may help guide research into ocular surface disease biomarkers and therapeutics. MethodsIn this prospective twin cohort study, healthy individuals were recruited from a population-based registry. Tear fluid was collected with the Schirmer test strips was submerged in phosphate buffered saline and stored at −80° before undergoing 27-cytokine multiplex immunoassay analysis. Broad-sense heritability (h2) of cytokine concentrations was analyzed. Results90 participants (23 monozygotic and 22 dizygotic twin pairs) were included. Data availability allowed for heritability analysis of 15 cytokines, and a h2 >50% was seen for 10 cytokines. A statistical power of >80% was achieved for heritability analyses of the cytokines interferon gamma induced protein 10 (h2 = 94.8%), eotaxin (89.8%), interleukin 7 (86.6%), interleukin 1β (82.2%) and monocyte chemoattractant protein 1 (68.2%). ConclusionsThe tear fluid concentration of several analyzed cytokines was found to be highly heritable. A considerable amount of the inter-individual variation observed for the concentration of certain tear fluid cytokines can be linked to hereditary factors that cannot easily be modified by changing factors in the environment of patients. This suggests that a higher success in ocular surface disease drug discovery may be anticipated for drugs that have targets in specific populations, and points to the importance of emphasizing known preventive measures of ocular surface disease and examinations of close relatives of patients with ocular surface disease, such as dry eye disease.

Evaluation of Dry Eye Severity and Ocular Surface Inflammation in Patients with Autoimmune Rheumatic Diseases

ABSTRACT Purpose To evaluate dry eye severity and ocular surface inflammation in autoimmune rheumatic diseases (ARDs). Methods Seventy-nine patients with ARDs were enrolled, including 26 patients with rheumatoid arthritis (RA), 33 patients with systemic lupus erythematosus (SLE), and 20 patients with primary Sjögren’s syndrome (pSS). All patients underwent ocular surface evaluations, including ocular surface symptoms, signs, conjunctival impression cytology, and tear multicytokine detection. Systemic conditions, including disease duration, disease activity, and serological parameters, were also noted. Results SLE patients had the shortest disease duration, and nearly half of them had low disease activity, while RA patients and pSS patients had a relatively long disease duration, and approximately 90% of them had moderate or high disease activity. The incidence of dry eye and the levels of the proinflammatory tear cytokines in SLE were significantly lower than those in RA and pSS. However, ocular surface squamous metaplasia was more severe in SLE and pSS than in RA. Dry eye severity in all ARD patients was shown to be independent of disease activity, while Nelson’s grades were positively correlated with disease duration in RA patients. Disease-related serological parameters were associated with tear proinflammatory cytokines in all ARD patients. Conclusions Variable degrees of dry eye and immune-mediated ocular surface inflammation persist in different ARD patients. In addition to a well-known association between dry eye and pSS, dry eye is also commonly observed in SLE and RA patients. Therefore, there is a definite need for regular ophthalmologic evaluations and topical medications in all patients with ARDs.

The Role of IL-6, IL-10, and TNF-α in Ocular GVHD Following Allogeneic Transplantation

ABSTRACT Purpose To figure out the roles of tear inflammatory cytokines in Ocular graft-versus-host disease (oGVHD) symptoms by analyzing tear cytokine levels and related factors. Methods This prospective study involved 27 post-HSCT patients and 19 controls with dry eye disease. Analyses included tear cytokine (IL-6, IL-10, and TNF-α), ocular surface evaluation, and conjunctival impression cell examination. Tear cytokine levels were evaluated in three grades of corneal epithelial lesions. The study also analyzed the correlation between tear cytokine levels and ocular surface parameters. Tear cytokine levels were then used in a Receiver Operating Characteristic (ROC) curve and linear regression model to predict oGVHD related factors. Results IL-6 has good diagnostic efficacy in oGVHD related dry eye. Elevated levels of tear IL-6 and TNF-α were observed in the group with severe corneal epithelial lesions. IL-6 levels were positively correlated with corneal fluorescein staining (CFS), eyelid margin hyperemia, conjunctival lesions, and meibum secretion. IL-6 showed excellent predictive ability with Area Under the Curve (AUC) values all greater than 0.70 (p < 0.05). IL-10 and TNF-α were negatively correlated with the meibomian gland proportion and conjunctival goblet cell (GC) density, while TNF-α was positively correlated with CFS and eyelid margin hyperemia. Conclusion Dry eye symptoms related to ocular GVHD, can be partly diagnosed and assessed using various tear cytokine level detection methods.