Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date, this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of eight healthy controls were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed. Resulting files were searched against the uniprot_swissprot database (2017_05_10) using Thermo Proteome Discoverer (version 2.2) and a false discovery rate of 1% was selected. Globally, 1351 proteins were identified with 2 unique peptides. More specifically, 39% of the lacrimal proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins emerged also as abundant lacrimal proteins. Pathway analyses highlighted among others the glycolysis and the coagulation and complement cascades. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

The aim was to investigate the levels of cytokines and soluble IL-6R in the tears of patients with thyroid-associated orbitopathy (TAO) disease. Schirmer’s test was adopted to collect tears from TAO patients (N = 20, 17 women, mean age (±SD): 46.0 years (±13.4)) and healthy subjects (N = 18, 10 women, 45.4 years (±18.7)). Lacrimal cytokines and soluble IL-6R (sIL-6R) were measured using a 10-plex panel (Meso Scale Discovery Company) and Invitrogen Human sIL-6R Elisa kit, respectively. Tear levels of IL-10, IL-12p70, IL-13, IL-6 and TNF-α appeared significantly higher in TAO patients than in healthy subjects. Interestingly, IL-10, IL-12p70 and IL-8 levels increased in tears whatever the form of TAO whereas IL-13, IL-6 and TNF-α levels were significantly elevated in inflammatory TAO patients, meaning with a clinical score activity (CAS) ≥ 3, compared to controls. Furthermore, only 3 cytokines were strongly positively correlated with CAS (IL-13 Spearman coeff. r: 0.703, p = 0.0005; IL-6 r: 0.553, p = 0.011; IL-8 r: 0.618, p = 0.004, respectively). Finally, tobacco use disturbed the levels of several cytokines, especially in patient suffering of TAO. The differential profile of lacrimal cytokines could be useful for the diagnosis of TAO patients. Nevertheless, the tobacco use of these patients should be taken into account in the interpretation of the cytokine levels.

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PurposeTo investigate the molecular composition of subretinal fluid (SRF) in central serous chorioretinopathy (CSCR) and rhegmatogenous retinal detachment (RRD) using proteomics and metabolomics.MethodsSRF was obtained from one patient with severe nonresolving bullous CSCR requiring surgical subretinal fibrin removal, and two patients with long-standing RRD. Proteins were trypsin-digested, labeled with Tandem-Mass-Tag and fractionated according to their isoelectric point for identification and quantification by tandem mass spectrometry. Independently, metabolites were extracted on cold methanol/ethanol, and identified by untargeted ultra-high performance liquid chromatography and high-resolution mass spectrometry. Bioinformatics analyses were conducted.ResultsIn total, 291 proteins and 651 metabolites were identified in SRF samples. Compared with RRD, 128 proteins (77 downregulated; 51 upregulated) and 76 metabolites (43 downregulated; 33 upregulated) differed in the SRF from CSCR. Protein and metabolites notably deregulated in CSCR were related to glycolysis/gluconeogenesis, inflammation (including serum amyloid P component, versican), alternative complement pathway (complement factor H and complement factor H–related protein), cellular adhesion, biliary acid metabolism (farnesoid X receptor/retinoid X receptor), and gluco- and mineralocorticoid systems (aldosterone, angiotensin, and corticosteroid-binding globulin).ConclusionsProteomics and metabolomics can be performed on SRF. A unique SRF sample from CSCR exhibited a distinct molecular profile compared with RRD.Translational RelevanceThis first comparative multiomics analysis of SRF improved the understanding of CSCR and RRD pathophysiology. It identified pathways potentially involved in the better photoreceptor preservation in CSCR, suggesting neuroprotective targets that will require additional confirmation.

PurposeThe tear film is a complex structure which constitutes an interface between our eyes and the external environment. Its protein, lipid and metabolite composition is highly regulated depending on several factors. Despite its attractive characteristics, this fluid is still poorly studied. Here we present an in‐depth human tear proteome based on a mass spectrometry approach.MethodsIn this study, tears of two healthy controls (two women aged 59 and 61 years) were collected with Schirmer's papers. After trypsin digestion and off‐gel electrophoresis fractionation, two proteomic analyses were performed by mass spectrometry (using LTQ Orbitrap Velos Pro coupled to a liquid chromatography). Resulting files were searched against the UniProt‐SwissProt/TrEMBL database (version 2014_10) and a false discovery rate of 1% was selected. The protein list was analysed using Ingenuity Pathways Analysis and Cytoscape software.ResultsGlobally 2105 and 825 proteins were identified with 1 and 2 unique peptides respectively after removing keratins and immunoglobulins. Regarding the 825 proteins, the top three pathways that we highlighted were the acute phase response signalling, the remodelling of epithelial adherens junctions and the clathrin‐mediated endocytosis signalling. Moreover, we identified 203 proteins that were not found in the previous published studies. By comparing our tears with others fluids, only 26.9% were identified in vitreous humor and 45.7% in plasma, confirming that tears have a specific composition.ConclusionsThanks to this study, we are able to propose an expanded tear proteome. Both specific proteins of the tears and correlations with other fluids give them a great potential for biomarker research.This study is kindly supported by the Provisu Foundation and the SNF_MVH (PMPDP3_158370).