This competitive renewal focuses on the regulation of ocular surface inflammation by lumican, an abundant extracellular matrix (ECM) protein at the ocular surface. Eight million people worldwide suffer from infections, allergies and autoimmune conditions leading to corneal scarring and blindness. The ECM has a significant role in modulating the local inflammatory milieu, while its breakdown can present immune cells with endogenous danger signals to drive excessive immune responses. However, the precise molecular mechanisms of ECM- immune interactions are not understood. In its most abundant form lumican is a structural protein that regulates collagen architecture to generate a transparent cornea. Recently, we identified its N-terminal domain to interact with leukocyte surfaces to promote toll-like receptor 4 (TLR4) mediated host response to gram-negative bacterial lipopolysaccharide (LPS) endotoxins. In a remodeling ECM during infections and inflammation, the immune-interactions of lumican can be a double-edged sword - needed for development of protective innate immunity, but unrestricted can drive chronic inflammation. Our current goals are to gain molecular insights into lumican - innate immune cell interactions, to develop strategies to strengthen protective immunity and diminish immune dysregulation using lumican-based peptides and antibodies. As our in vivo inflammatory setting we will use LPS and Pseudomonas aeruginosa (PA) keratitis models in wild type and lumican-deficient mice. Additional recombinant lumican variants, peptides, anti-lumican antibodies and cell cultures models have all been developed in the laboratory for this study. The following hypotheses will be tested in three aims.
Aim 1 : Hypothesis - Lumican promotes immune response by increasing pericellular LPS or modulates lipid rafts to enhance TLR4 recruitment to the cell surface. Lipid rafts are centrally important in innate immune signal transduction and clinically relevant as targets of therapy. We will use primary macrophages, cell lines, recombinant lumican, biochemical, confocal and FRET analyses to address lumican involvements at the cell surface.
Aim 2 : Hypothesis - The immunologically active N-terminal domain is unavailable for immune cell interactions in collagen-associated lumican. We will use recombinant lumican and three variants that either have the N-terminal domain or the central collagen binding domain to test this hypothesis. Collagen-pretreated lumican variants will be tested for their abilities to induced LPS response in culture. Secondly, the variants will be transiently expressed at the ocular surface of wild type and lumican deficient mice, and challenged with keratitis to address their role in modulating the cytokine milieu, innate and adaptive immune cells and ocular surface inflammation.
Aim 3 : Hypothesis - Ocular surface inflammation can be modulated by lumican-based peptides or anti-lumican antibodies. We will test if an antibody against the N- terminal domain or mimetic peptides can modulate lumican-immune cell interactions to ameliorate inflammation in the keratitis models to develop the premises for localized treatments of inflammation.

Public Health Relevance

Most treatments of inflammation due to infections autoimmune dry eye diseases involve strong antimicrobial agents or immunosuppressants with harmful side effects. Our findings on immunomodulatory roles of lumican will lead to future alternative strategies for milder localized treatments of inflammation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
3R01EY011654-17A1S1
Application #
8919487
Study Section
Special Emphasis Panel (DPVS)
Program Officer
Mckie, George Ann
Project Start
1997-01-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
17
Fiscal Year
2014
Total Cost
$47,609
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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