NET formation by neutrophils

NETs, which are generated during a process termed NETosis, represent the most
recently described weapon in the arsenal of neutrophils to combat pathogens. In
the process of NETosis, the neutrophil ejects its chromatin, which is decorated
with antimicrobial proteins. The resulting neutrophil extracellular trap (NET)
is a web-like structure composed of DNA and proteins that can catch invading
pathogens and prevent them from spreading through the rest of the body. The
formation of NETs was described for the first time in 2004, and since then a
lot of research has been performed to obtain more insight in the process of NET
formation, although still many questions remain unanswered. With the current
study we would like to increase our fundamental knowledge of NET formation.

NETosis can be well studied in in vitro experiments, using (human) neutrophils
isolated from freshly drawn blood of healthy individuals. The isolated
neutrophils are seeded in plastic well-plates or petri-dishes and subsequently
stimulated with different inducers of NETosis. In the literature, various
stimuli have been described and because these are not uniformly used by
different research groups it is often hard to compare existing data.
Importantly, all these stimuli appear to induce NETosis via distinct
mechanisms. We aim to shed more light on the diversity of the corresponding
pathways and the cellular and molecular features that characterize them.

The characterization of NET formation is clinically relevant since NETosis has
been implicated in many diseases since its first description in 2004. It was
soon anticipated that the exposure of the immune system to DNA and other
intracellular components might lead to an autoimmune response. Subsequently,
several investigations have addressed the influence of NETs on autoimmune
diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis
(RA). Furthermore, the antimicrobial proteins associated with NETs may also be
harmful to host tissue if not properly cleared. Finally, NETs were shown to
obstruct blood vessels and induce clotting, and might therefore also be
involved in thrombotic diseases. A better fundamental understanding of NETosis
might aid in the development of treatment for the above-mentioned disease
states, for example by designing drugs that inhibit NET formation, or drugs
that efficiently degrade NETs to prohibit clogging and tissue injury.

To gain a better understanding of NET formation by human neutrophils and the
molecular mechanisms underlying this process.
We would like to gain insight in:
* The differences in NET formation upon the exposure of neutrophils to
different stimuli.
* The involvement of protein-modifying enzymes in NET formation
* The citrullinated protein content of NETs
* The intracellular calcium and ROS levels during NET formation

This study is based on the isolation of neutrophils from blood donated by
healthy volunteers. NET formation will be studied in in vitro experiments.
Blood samples will be collected via venipuncture after signed informed consent;
the volume will be between 10 and 40ml. The exact amount depends on the nature
of the planned experiment and the amount of neutrophils that is needed to
perform it. Donors are not followed over time or subjected to other procedures
than donating blood. This research will not yield findings that maybe stressful
or clinically relevant to the donors since the only clinical parameter that
will be checked are cell counts. Since these cell counts are different anyway
between different donors, no meaningful conclusions can be drawn upon the
outcome of these numbers. This study does not entail minors or incapacitated
adults.

We have chosen for this study design instead of purchasing so-called buffy
coats of Sanquin, since neutrophils only live for one day and products of
Sanquin are often stored overnight. Moreover, we only need relatively small
amounts of blood to be able to perform experiments. For reliable NET formation
experiments, it is important to collect the material via standardized
procedures, which can be best controlled when the blood is obtained as freshly
as possible.

After oral informed consent, the healthy donor will undergo venipuncture to
donate 10 to 40 ml blood, which is collected in EDTA anti-coagulation tubes.
The venipuncture is performed by nurses from the departments of Neurology at
the RadboudUMC. Neutrophils are isolated from the donated blood according to
Standard Operating Procedures and subsequent in vitro experiments will be
performed on the day of donation. After the experiments are finished, all
materials will be discarded; biomaterials involved in this study will not be
stored.

Risks and burdens: Risk is negligible and the burden is minimal. The most
common risks related to drawing blood from the subject*s arm are brief pain
and/or bruising. Infection, excess bleeding, clotting or fainting is also
possible but unlikely.
Benefits: There will not be direct benefit. However, participation of subjects
can help researchers to gain knowledge.