Lipid mediators and their signaling in ocular surface inflammation and meibomian gland dysfunction

Dry eye (DE) affects 1 in 5 veterans and impacts quality of life. It is a complex disease that manifests with multiple symptoms (e.g. pain, visual disturbance) and signs (e.g. decreased tear production, increased evaporation). Key pathophysiological components of DE are ocular surface inflammation and meibomian gland dysfunction (MGD). Despite its frequency and morbidity, current therapies do not relieve symptoms in all patients. In order to appropriately manage DE, it is necessary to understand mechanisms and specific contributors to disease sub-types. The overall goal of this proposal is to improve such knowledge by studying the role of bioactive sphingolipids (SPL) in MGD and other aspects of DE. Our preliminary and published work suggest SPL composition changes (higher sphingomyelin (SM)/ ceramide (Cer) ratio) in poor quality meibomian gland secretion (meibum); a higher ratio of pro-inflammatory (ω-6) /pro-resolving (ω-3) lipids, and higher levels of prostaglandin E2 in tears from DE patients. Bioactive Cer generated from SM by sphingomyelinases (SMase) and its derivatives, ceramide 1-phosphate and sphingosine 1-phosphate are inflammatory lipids and can induce formation of other inflammatory lipids such as prostaglandins. We detected SMase activity in human tears. In this proposal, we will test the hypothesis that changes in SPL composition in meibum contribute to various signs of DE including ocular surface inflammation, tear film disturbances, and MGD. In Aim 1, we will profile meibum and tear SPL and pro- and anti-inflammatory PUFAs and eicosanoids by LC-MS/MS from 120 cases (poor meibum quality) and 120 controls (normal meibum quality), chosen from our prospectively collected samples from a large veteran population after a comprehensive eye evaluation (mean age 62±10; 467 males, 46 females; 230 white, 272 black). Correlation and regression analysis will be performed with clinical data as dependent variables and lipid mediators as independent variables. In Aim 2, we will determine acitivity of SMAse (acidic and neutral) from 240 Schirmer's strips corresponding to the samples in Aim 1. We will then build models looking at relationships between SMase activity, SPL, and ocular surface inflammation. In Aim 3, we will identify molecular connections between SPL and inflammation in an in vitro model by using human immortalized meibomian gland epithelial cell line (HMGEC) to test the effects of bioactive SPL on mediating inflammation, cell viability, cell death etc. in normal and hyperosmolar conditions. Impact: Our results will identify SPL differences by MGD status, elucidate relationships between bioactive SPL, inflammatory lipid mediators, and SMase activity with ocular surface inflammation and clinical features of MGD and DE sub-types. The knowledge generated will advance the field by determining the relative contributions of different lipids on different manifestations of DE and provide the foundation for developing new molecular targets for therapy.