Download Project name: Release of Neutrophil Extracellular Traps (NETs) in

Project name: Release of Neutrophil Extracellular Traps (NETs) in response to
inflammatory stimuli
Instructor: Slava Berger, Department of Medicine
Abstract/introduction:
Neutrophils are the major antimicrobial phagocytes of the innate immune system.
They are one of the first effector cells to arrive at the site of infection and play
critical roles in pathogen clearance, recruitment, as well as in activation of other
immune cells. To combat microbes, neutrophils employ three major strategies: the
well-known (1) phagocytosis and (2) degranulation, which are beyond the scope of
this research; (3) A novel host defense structure named neutrophil extracellular
traps (NETs).
NETs are networks of DNA and antimicrobial factors that are released by
neutrophils to trap and kill pathogens. They can trap and kill various bacterial,
fungal and protozoan pathogens, and their release is one of the first lines of
defense against pathogens.
NETs may act serving as effective antimicrobial defenses, but also as putative
sources of molecules that induce inflammation and may promote tissue damage.
Deficient NET formation predisposes humans to severe infection, while
uncontrolled NET formation contributes to inflammation, vascular injury and tissue
damage. Making too many or not disposing of NETs at the right time and in the
right place is pathogenic. Therefore, regulating NET formation is important to
prevent many pathological conditions. Thus the present study will determine the
effect of various inflammatory and pathogenic stimuli on the release of NETs.
In this project we will isolate human neutrophils from the blood of healthy
volunteers. We will grow the cells in culture and activate them with various
inflammatory and pathogenic stimuli. These stimuli will include pro-inflammatory
molecules, membrane components from bacterial and fungal origin. After the
stimulation the morphological changes of the cells will be followed by light
microscopy. NET formation will be visualized by staining with specific antibodies
and fluorescent dyes, and will be quantified. The expected results will contribute to
further research of NET formation phenomenon in various pathologies and health
condition.
Figure 1. Bacteria caught in NETs. Scanning electron microscopy of human neutrophils incubated with Salmonella, a
bacterium that causes typhoid fever and gastroenteritis. The bacteria are trapped in NETs. Bar, 1 μm (Brinkmann et al. J
Cell Biol. 2012, 198:773-83).
Figure 2. Visualizing NETs using neutrophil antimicrobial antibodies or DNA-intercalating dyes by confocal microscopy.
Image was taken in our laboratory.
Student mission / Objective:
1. Acquiring knowledge and being exposed to the work with human blood and
leukocyte isolation using Ficoll-gradient.
2. Flow Cytometry analysis of various leukocyte populations using
specific antibodies.
3. Working with neutrophil primary cell cultures.
4. Studying the NET formation by using fluorescent staining of the cells and analysis
with flourometer and confocal microscopy.
5. Studying proteins which mediate NET formation using confocal microscopy and Western
blotting analysis.
6. Observation of NET formation using live-cell imaging Time-Lapse microscopy.
Requirements:
1. Basic background on the inflammatory/immune system
2. Basic knowledge of human blood and its structure.
3. The willingness to work with human blood and
cells
Please read the attached paper entitled: “Neutrophil extracellular traps: Is immunity the
second function of chromatin?” and answer the following questions:
1. Write a brief summary about the innate immune system and its function.
2. Name the blood cells and write the main role of each cell.
3. Write a brief summary about the role of NET formation in one of the diseases
mentioned in the attached paper.
Recommended reading material:
In order to answer these questions, you are free to use scientific papers and scientific
websites including Wikipedia.
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If you have any questions or trouble understanding the requirements, feel free to
contact me : [email protected]