Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

点击翻译按钮获取中文摘要

Project Summary Although 15-20% of Americans suffer from allergic conjunctivitis, current treatments are limited to anti- histamines and mast cell stabilizers that are often ineffective. The long-term goal of the proposal is to develop a new treatment for allergic conjunctivitis that could also be used for allergies of the lung, skin, and nose along with other inflammatory diseases of the ocular surface such as dry eye. The new treatment is an anti- inflammatory and pro-resolution lipid mediator, the D-series resolvins compared to E-series resolvins currently in a clinical trial. Conjunctival goblet cell oversecretion of mucous including the mucin MUC5AC is one of the symptoms of allergic conjunctivitis that include itching, conjunctival redness, tearing, and chemosis. D-, but not E-, series resolvins have two modes of action. They block histamine and leukotriene over stimulation of goblet cell secretion and themselves activate a small amount of secretion to protect the ocular surface while the over secretion is being reset. E-series do not themselves increase secretion. The current proposal will focus on determining how pro-resolution mediators act at a molecular level to counter-regulate proinflammatory mediator stimulation of goblet cell mucin secretion both in culture and in vivo in a mouse model of allergic conjunctivitis. Research will focus on the following aims: 1. Determine the cellular mechanisms the D- and E- series resolvins use to stimulate goblet cell mucin secretion and if goblet cells are the source of the resolvins and other lipid mediators; 2. Unravel the molecular mechanisms D- and E-series resolvins use to activate protein kinases to interact with histamine and leukotriene receptors to counter-regulate their action; and 3. Ascertain the efficacy of D- compared to E-series resolvins to terminate goblet cell secretion and improve symptoms in a murine model of allergic conjunctivitis. For aim 1 RvD1, aspirin triggered (AT)-RvD1, and RvE1 will be used to stimulate human conjunctival goblet cells in culture. The receptors and cellular signaling pathways activated by the resolvins including phospholipase C and D and PI-3K will be investigated using siRNA, pharmacological inhibitors, and adenovirus constructs. In addition the production of pro-inflammatory and pro-resolution mediators will be determined by lipidomic measurements on goblet cells in culture. For aim 2 CHO cells transfected with a pro-inflammatory histamine or leukotriene receptor and a pro-resolution receptor and cultured human goblet cells will be used. Protein kinases predicted to phosphorylate the pro- inflammatory mediator receptors will be studied to determine if RvD1, AT-RvD1, and RvE1 activate the protein kinases and counter-regulate the histamine or leukotriene receptor to block its activity and terminate mucin secretion. siRNA, pharmacological inhibitors, and adenovirus constructs will be used for this aim. For aim 3 a mouse model of severe allergic conjunctivitis will be used with ovalbumin as the allergen. Resolvins compared to anti-histamines will be used to determine if they decrease MUC5AC secretion and goblet cell proliferation, ameliorate the clinical symptoms of allergic conjunctivitis, and terminate infiltration of inflammatory cells.