Clinical proteomics elucidated the complex molecular mechanisms in the progression of dry eye syndrome with different severity levels: a step forward to personalized diagnosis

Dry eye syndrome (DES) is a collective term used to define a pathology of the ocular surface with pleiotropic etiology that affects the tear film homeostasis and results in perturbations in the visual function. However, specific clinical biomarkers associated with the vicious cycle involving multiple interacting pathways that perpetuate and exacerbate the progression of DES are still largely lacking. Therefore, this study was undertaken to unravel the intricate proteome changes in individuals with increasing severity of different DES subgroups.

Subjects underwent a battery of clinical examinations including the basic secretory test, tear break-up time and ocular surface disease index. Subsequently, the subjects were divided into evaporative (DRYlip) and aqueous-deficient (DRYaq), as well as the combination of both DES (DRYaqlip) with increasing severity (normal, moderate and severe) and healthy (CTRL) based on distinct clinical attributes. Next, the individual tear samples (N=322) were analyzed employing a rapid and robust mass spectrometry (MS)-based proteomics strategy, followed by bioinformatics analyses to elucidate the underlying molecular processes and protein functionalities.

A total of 71, 218 and 306 proteins were found to be significantly (p < 0.05) differentially regulated in DRYlip, DRYaq and DRYaqlip vs. CTRL, respectively. The hallmark of this study was the identification of 30 lacrimal gland-specific proteins comprising mainly LTF, LYZ, LACRT and PROL1 secreted primarily via the exocytotic pathway, which were found to be progressively down-regulated in DRYaq and DRYaqlip vs. CTRL (e.g. LTF: moderate = Δlog₂ -1.8; severe = Δlog₂ -3.4). Inflammatory response was found to be increasingly activated in DRYlip (p=3.8×10^-4), DRYaq (p=4.2×10^-9) and DRYaqlip (p=2.1×10^-11). Likewise, proteins involved in apoptosis (p=1.0×10^-15), antibacterial response (p=2.3×10^-5) and synthesis of reactive oxygen species (p=1.3×10^-12) were also found to be increasingly activated in all three DES subgroups. Notably, glycolysis (p=2.6×10^-8), actin cytoskeleton signaling (p=1.0×10^-3) and complement system (p=4.5×10^-6) were found to be activated only in patients with moderate and severe DRYaqlip.

In summary, our study took advantage of the MS-based proteomics strategy to analyze the largest number of tear samples in a clinical setting to date, considering different DES subgroups and varying levels of disease severity. Notably, this study has demonstrated that the protein markers involved in the exocytotic pathway, which play major roles in the production of tears, were significantly impaired. This resulted in the gradual activation of the vicious cycle involving the interplay between various pathways in the progression of DES. Importantly, the key clinical protein markers for DES were unraveled for the first time and could be potentially used for improved clinical diagnosis and targeted therapeutic interventions in ameliorating DES in a personalized manner.