The role of the Tear Film and Ocular Surface Society (TFOS) Dry Eye Workshop (DEWS) II Diagnostic Methodology Subcommittee was 1) to identify tests used to diagnose and monitor dry eye disease (DED), 2) to identify those most appropriate to fulfil the definition of DED and its sub-classifications, 3) to propose the most appropriate order and technique to conduct these tests in a clinical setting, and 4) to provide a differential diagnosis for DED and distinguish conditions where DED is a comorbidity. Prior to diagnosis, it is important to exclude conditions that can mimic DED with the aid of triaging questions. Symptom screening with the DEQ-5 or OSDI confirms that a patient might have DED and triggers the conduct of diagnostic tests of (ideally non-invasive) breakup time, osmolarity and ocular surface staining with fluorescein and lissamine green (observing the cornea, conjunctiva and eyelid margin). Meibomian gland dysfunction, lipid thickness/dynamics and tear volume assessment and their severity allow sub-classification of DED (as predominantly evaporative or aqueous deficient) which informs the management of DED. Videos of these diagnostic and sub-classification techniques are available on the TFOS website. It is envisaged that the identification of the key tests to diagnose and monitor DED and its sub-classifications will inform future epidemiological studies and management clinical trials, improving comparability, and enabling identification of the sub-classification of DED in which different management strategies are most efficacious.

TFOS DEWS II Diagnostic Methodology report

Biomarkers provide a powerful and dynamic approach to improve our understanding of the mechanisms underlying ocular diseases with applications in diagnosis, disease modulation or for predicting and monitoring of clinical response to treatment. Defined as measurable indicator of normal or pathological processes, biomarker evaluation has been used extensively in drug development within clinical settings to better comprehend effectiveness of treatment in ocular diseases. Biomarkers in the eye have the advantage of access to multiple ocular matrices via minimally invasive methods. Repeat sampling for biomarker assessment has enabled reproducible objective measures of disease process or biological responses to a drug treatment. This review describes the usage of biomarkers with respect to four commonly sampled ocular matrices in clinic: tears, conjunctiva, aqueous humor and vitreous. Issues that affect the evaluation of biomarkers are discussed along with opportunities to leverage biomarkers such that ultimately, they can be used for customized targeted therapy.

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Review of Biomarkers in Ocular Matrices: Challenges and Opportunities.

Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-β), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

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Immune Privilege and Eye-Derived T-Regulatory Cells

Th17 cells are critical effectors mediating the ocular surface autoimmunity in dry eye disease (DED). Increased IFN-γ has also been implicated in DED; however, it remains unclear to what extent Th1 cells contribute to DED pathogenesis. In this study, we investigated the cellular source of IFN-γ and assessed its contribution to corneal epitheliopathy in DED mice. We discovered a significant IL-17A+IFN-γ+ (Th17/1) population and determined that these cells are derived from Th17 precursors. Adoptive transfer of Th17/1, but not Th1, cells confers the disease to naive recipients as effectively as do Th17 cells alone. DED-induced IL-12 and IL-23 are required for in vivo transition of pathogenic Th17 cells to IFN-γ producers. Furthermore, using IFN-γ-deficient Th17 cells, we demonstrate the disease-amplifying role of Th17-derived IFN-γ in DED pathogenesis. These results clearly demonstrate that Th17 cells mediate ocular surface autoimmunity through both IL-17A and IFN-γ.

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IFN-γ-Expressing Th17 Cells Are Required for Development of Severe Ocular Surface Autoimmunity.

Purpose Biomarkers with minimally invasive and reproducible objective metrics provide the key to future paradigm shifts in understanding of the underlying causes of dry eye disease (DED) and approaches to treatment of DED. We review biomarkers and their validity in providing objective metrics for DED clinical research and patient care. Methods The English-language literature in PubMed primarily over the last decade was surveyed for studies related to identification of biomarkers of DED: (1) inflammation, (2) point-of-care, (3) ocular imaging, and (4) genetics. Relevant studies in each group were individually evaluated for (1) methodological and analytical details, (2) data and concordance with other similar studies, and (3) potential to serve as validated biomarkers with objective metrics. Results Significant work has been done to identify biomarkers for DED clinical trials and for patient care. Interstudy variation among studies dealing with the same biomarker type was high. This could be attributed to biologic variations and/or differences in processing, and data analysis. Correlation with other signs and symptoms of DED was not always clear or present. Conclusions Many of the biomarkers reviewed show the potential to serve as validated and objective metrics for clinical research and patient care in DED. Interstudy variation for a given biomarker emphasizes the need for detailed reporting of study methodology, including information on subject characteristics, quality control, processing, and analysis methods to optimize development of nonsubjective metrics. Biomarker development offers a rich opportunity to significantly move forward clinical research and patient care in DED. Overview DED is an unmet medical need - a chronic pain syndrome associated with variable vision that affects quality of life, is common with advancing age, interferes with the comfortable use of contact lenses, and can diminish results of eye surgeries, such as cataract extraction, LASIK, and glaucoma procedures. It is a worldwide medical challenge with a prevalence rate ranging from 8% to 50%. Many clinicians and researchers across the globe are searching for better answers to understand the mechanisms related to the development and chronicity of DED. Though there have been many clinical trials for DED, few new treatments have emerged over the last decade. Biomarkers may provide the needed breakthrough to propel our understanding of DED to the next level and the potential to realize our goal of truly personalized medicine based on scientific evidence. Clinical trials and research on DED have suffered from the lack of validated biomarkers and less than objective and reproducible endpoints. Current work on biomarkers has provided the groundwork to move forward. This review highlights primarily ocular biomarkers that have been investigated for use in DED, discusses the methodologic outcomes in providing objective metrics for clinical research, and suggests recommendations for further work.

The Growing Need for Validated Biomarkers and Endpoints for Dry Eye Clinical Research.

Purpose
Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups.

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Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods.

PURPOSE The purpose of this study is to explore the role of the adaptive immune system in patients with aqueous-deficient dry eye (ADDE) and lipid-deficient dry eye (LDDE). METHODS Patients (n = 29) with moderate to severe dry eye (dry eye workshop [DEWS] severity grading scheme) were enrolled in a cross-sectional study and classified as ADDE (Schirmer < 10), LDDE (abnormal meibum), combined (meeting both criteria), or generic (meeting neither criterion). Tears were collected by Schirmer strips, and samples for both eyes were pooled for each subject. Thirty micrograms of total protein was used in a normalized volume for microarray analysis (Quantibody Human Inflammation Array 3; RayBiotech). Six markers of TH1 cells (interferon [IFN]γ, interleukin [IL]-2), TH2 cells (IL-4, IL-5, IL-13), and TH17 cells (IL-17) were assessed. RESULTS ADDE demonstrated the highest total cytokine concentration, followed by the LDDE, combined, and generic groups. IFNγ and IL-2 were detectable in all subgroups. IL-4, -5, and -13 were detectable in ADDE and LDDE, but only IL-13 was detected in both the combined and generic groups. IL-17 was present in the ADDE, LDDE, and combined groups. CONCLUSIONS TH1 cells seem to be involved in all forms of dry eye. ADDE and LDDE seem to be mediated by TH1, TH2, and TH17 cells. The combined-mechanism group is mediated by TH1 and TH17 cells, and generic dry eye seems to be mediated by TH1 cells only. ADDE has the greatest overall T-cell-mediated pathophysiology compared with the other subgroups, which is consistent with previous reports of improved efficacy with antiinflammatory therapy in these patients.

Karen Dionne

Papers

Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods.

Differential Profiling of T-Cell Cytokines as Measured by Protein Microarray Across Dry Eye Subgroups.

The Impact of Treatment on 13-Cis Retinoic Acid-Challenged Human Meibomian Gland Epithelial Cells