Neutrophils are the early responders to acute inflammation during vessel wall injury or insult. Apart from phagocytosis and degranulation, NETosis (formation of Neutrophil Extracellular Trap or NET) is another aspect of neutrophil-mediated defense mechanism. Multiple proteins/enzymes such as histones, neutrophil elastase and myeloperoxidase are released along with NETs. Therefore, induction of NETosis in sterile inflammation is deleterious for many chronic diseases such as atherosclerosis. Abdominal aortic aneurysms (AAAs) remain a life-threatening disease in US adults with no available nonsurgical therapies highlighting a great need to develop a treatment strategy. During AAA formation, aortic wall undergoes major pathological changes with degradation of extracellular matrix and marked infiltration of inflammatory cells, such as neutrophils and lymphocytes. In murine experimental models, neutrophils have been detected in early stages of AAA growth. Recently, we have shown that genetic (IL-1?? and IL-1R1 deficiency in mice) and pharmacological (anakinra treatment) inhibition of IL-1?? signaling protect mice from AAA formation with significant reduction in aortic infiltration of inflammatory cells including neutrophils. Our preliminary result and published literatures suggest that IL-1?? induces NETosis in neutrophils. Therefore, we hypothesize that aortic IL-1? induces NETosis and exacerbates inflammation in AAA. To test this hypothesis, we are proposing three specific aims. First we will test if genetic and pharmacological inhibition of IL-1? signaling suppress NETosis and the AAA growth in mice. For these aims, we will use C57BL/6 (wild-type, WT), IL-1?? knockout (KO) and IL-1R1 KO mice, mouse AAA models such as elastase perfusion and angiotensin II infusion, knockdown IL-1?? or IL-1R1 using AMAXA Nucleofector, adoptive transfer of IL-1R1 KO neutrophils to neutrophil depleted WT mice. To examine and quantify NETosis, we will use the state-of-the-arts immunohistochemistry, confocal and intravital microscopy, and imaging flow cytometry. We will show that NET formation promotes inflammation and AAA growths, and, pharmacological inhibition of NETosis by (i) impairing IL-1?? signaling (using anakinra) and (ii) degrading NETs (using DNase), suppresses AAA growth in mice. We will further test the hypothesis that human neutrophils exhibit IL-1?? -dependent NETosis and induce inflammation in human aortic wall ex vivo. We will use proteomics approaches to identify proinflammatory proteins in human NETs, use human aortic explants to examine inflammatory response of IL-1b-induced NETs and parallel flow chambers to examine IL-1?-induced NET-mediated immune cell recruitment from blood. Altogether, this proposal includes innovative concept that IL-1?? synthesized in aortic wall induces NETosis leading to increased tissue inflammation and AAA growth, and innovative methods to reduce NETosis and suppress AAA growth.
These aims when achieved will provide the foundation for developing novel therapies for AAAs in human.

Public Health Relevance

Abdominal aortic aneurysm (AAA) is a life-threatening disease characterized by ballooning of the abdominal aorta. At present there are no medical therapies therefore they can only be treated by surgery. In the line of developing a non-surgical method, we are proposing to understand and suppress the harmful effects of a particular cell type of the immune system called neutrophils, which are thought to initiate and promote the growth of AAA. Upon completion of the proposed research work, we will be able to show how neutrophils regulate AAA growth and we will be able to provide a putative treatment strategy for this disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01HL124131-01
Application #
8765843
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Tolunay, Eser
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Virginia
Department
Surgery
Type
Schools of Medicine
DUNS #
City
Charlottesville
State
VA
Country
United States
Zip Code
22904