P. aeruginosa (PA) frequently infects immunocompromised individuals with HIV-1, cancer, and cystic fibrosis (CF). Since PA is increasingly resistant to antibiotics, its infection often leads to either severe states or chronic situations with a persistent inflammatory response. Better understanding of host-pathogen interaction may suggest a more effective approach to combating this pathogen. MCP-1 is a major chemokine secreted by alveolar epithelial cells type II (AECII). Recent research has illustrated an immune role of AECII in PA infection, but the underlying mechanism remains unidentified. Our long-term goal is to understand the mechanism of host immunity and develop new strategies for controlling respiratory infections. The objective of this application is to elucidate the immune function of AECII, in particular through their secretion of cytokines and activation of AM. Our central hypothesis is that AECII can secrete cytokines (MCP-1) to enhance AM's anti-bacterial immunity through a lipid raft- mediated mechanism. We have formulated this hypothesis based on our recent findings that both AECII and AM participate in innate immunity against PA. We further found that membrane lipid rafts may be instrumental for regulating cytokine secretion. Using our primary cell model, we have discovered an immune role of AECII in enhancing AM's immunity using a conditioned AECII medium. Our data also suggest that AECII play a critical role in PA infection by secreting MCP-1 and recruiting the classically activated macrophages (CAM). The rationale is that elucidating how AECII enhance AM immunity will indicate a potential strategy to bolster immunity against PA. Our laboratory is ideally suited for this research, having the relevant expertise in isolation and culture of AECII a well as in lung infection models. We propose the following three specific aims:
Specific Aim 1 : Define the immune role of AECII cells in secreting cytokines during PA infection. We will identify the source of MCP-1 using in situ hybridization with AECII marker SPC. We will also use primary AECII culture to show MCP-1 as a dominant cytokine. Furthermore, AM and AECII from MCP-1-/- mice will be examined for their decreased immune function against PA infection. We will determine the ability of AECII in recruiting the classically activated macrophages (CAM).
Specific Aim 2 : Evaluate how lipid rafts regulate MCP-1 secretion in AECII. We will study the underlying mechanism for MCP-1 secretion and hopefully identify the involvement of ceramide-rich membrane microdomains. Acid shingomyelinase will be blocked by siRNA and chemical inhibitors for analyzing sphingolipid hydrolysis during PA early infection.
Specific Aim 3 : Assess the potential of super-AECII over-expressing MCP-1 in enhancing anti-PA capacity of human AM. We will create super-AECII using retroviral vectors to secrete high levels of MCP-1 and test their host defense in PA infection. We will also demonstrate that human AM can be activated by AECII and that this translational research may imply the clinical value of the immune AECII. This research will be performed by graduate and undergraduate students. Our efforts are expected to substantially advance understanding of this previously unrecognized immune function of AECII in activating AM, and may provide insights into mechanisms of cytokine secretion, with indications in development of novel therapeutics for treating this infection.

Public Health Relevance

P. aeruginosa (PA) is a bacterium that causes severe infections, particularly in immunodeficient individuals who are suffering tuberculosis, cancer, AIDS, severe burns, and cystic fibrosis. Because PA is increasingly resistant to antibiotics, its infection usually leads to a chronic state of persistent inflammatory response. We have made the surprising discovery that MCP-1, a versatile cytokine from alveolar epithelial cells, regulates host defense and inflammatory response in PA infection. We have also noted that lipid rafts may be important for regulating cytokine production. Through secretion of MCP-1, the alveolar epithelial cells may recruit a particular subset of macrophages (i.e., classically activated macrophages) to promptly respond to infection. Studying the immune role of alveolar epithelial cells may provide new insights into the development of novel treatment for PA infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15AI101973-01
Application #
8367586
Study Section
Special Emphasis Panel (ZRG1-IMM-E (90))
Program Officer
Taylor, Christopher E,
Project Start
2012-05-16
Project End
2015-04-30
Budget Start
2012-05-16
Budget End
2015-04-30
Support Year
1
Fiscal Year
2012
Total Cost
$414,000
Indirect Cost
$114,000
Name
University of North Dakota
Department
Biochemistry
Type
Schools of Medicine
DUNS #
102280781
City
Grand Forks
State
ND
Country
United States
Zip Code
58202
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