Bacterial conjunctivitis and keratitis occur in approximately 25,000 Americans annually with Staphylococcus aureus (S. aureus) being the leading cause of infection. If untreated, corneal infections in particular can be sight threatening In addition, the emergence of methicillin-resistant S. aureus makes development of new approaches to control ocular surface infections an immediate goal. Corneal response to infection is constrained, but conjunctiva can respond exuberantly. In particular the MUC5AC-secreting conjunctival goblet cells are the first line of innate immune defense of the ocular surface with their secreted mucin (MUC5AC) trapping and removing the bacteria. It is not known, however, if bacteria interact with goblet cells. The long-term object of this project is to determine: a) if bacteria interact directly with conjunctival goblet cells, b) what cellular signalng mechanisms and functions are triggered in the goblet cells, c) if activation of these functions prevents bacterial keratitis and conjunctivitis, and d) if drugs that activate these functions can e developed to treat ocular surface infections. An innovative hypothesis is that bacteria interact with conjunctival goblet cells and stimulate two distinct responses. First, goblet cells secrete mucin to trap and remove bacteria. Second, goblet cells activate the newly discovered Nod-like receptor (NLRP) 3 an intracellular responder to bacteria. Activation of NLRP3 causes formation of a multi-component complex, the inflammasome that result in the secretion of mature IL-1b that initiates innate mediated inflammation. A second component of this hypothesis is that pathogenic, but not commensal, bacteria interact differently with the goblet cells so that goblet cells mount an immune response against pathogenic, but not commensal, bacteria. The following specific aims will be investigated: 1) Does interaction with goblet cells by pathogenic toxin-forming S. aureus, but not by commensal, non-toxigenic S. epidermidis, cause a protective response by stimulating goblet cell mucin secretion and are the secretory responses distinct because the two types of bacteria activate diverse Toll-like receptor (TLR) dimers (TLR2/1 versus TLR2/6) and different signaling pathways ([Ca2+]/extracellular regulated-kinase (ERK)1/2 versus phosphatidylinositol-3 kinase (PI-3K).AKT)? and 2) Does interaction of pathogenic S. aureus, but not of commensal S. epidermidis, activate the goblet cell NLRP3 inflammasome to produce mature IL-1b and is the mechanism activated: a) channel formation or b) production of reactive oxygen species (ROS),? Cultured rat and human conjunctival goblet cells will be incubated with bacteria or lipoproteins that are pathogen-associated molecular patterns (PAMPs). Intracellular [Ca2+] will be measured by fluorescence microscopy, ERK1/2 by western blotting, and mucin secretion by biochemical assay. NLRP3 formation will be investigated by immunoprecipitation and western blotting, NLRP3 activation by FLICA assay, and secretion of mature IL-1b by ELISA. Agonists, antagonists, chemical inhibitors, and siRNA will be used to characterize responses.

Public Health Relevance

Bacterial conjunctivitis and keratitis occur in approximately 25,000 Americans annually. If untreated, they can be sight threatening. Furthermore, antibiotic resistant strains of bacteria have emerged, making these infections resistant to first line treatments. Therefore, developing new approaches to control ocular surface infection is an important goal.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY022415-02
Application #
8461558
Study Section
Special Emphasis Panel (ZRG1-BDCN-H (02))
Program Officer
Mckie, George Ann
Project Start
2012-05-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
2
Fiscal Year
2013
Total Cost
$414,675
Indirect Cost
$200,925
Name
Schepens Eye Research Institute
Department
Type
DUNS #
073826000
City
Boston
State
MA
Country
United States
Zip Code
02114
Dartt, Darlene A; Masli, Sharmila (2014) Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation. Curr Opin Allergy Clin Immunol 14:464-70