Neurology, Stroke Research (Liesz)

Project description

Stroke is known to induce pronounced neuroinflammation and infiltration of peripheral immune cells^4,5 . Besides the local inflammatory responses in the brain, stroke does also alter the peripheral immune homeostasis^6-8. Although neuroinflammation and the role of different cell types are well described, the understanding of peripheral immune alterations needs further investigations. To date, it is known that the peripheral immune alterations appear to be multiphasic. An acute immune activation⁷ can cause subacute immunosuppression⁹ and a state of chronic immune alterations challenging comorbidities, such as atherosclerosis8. It is of importance to have reliable surrogate marker to prevent these severe immune reactions, which increase the susceptibility for infections and risk of recurrent ischemic events in stroke patients. Our recent work9 and complementary studies¹⁰ revealed that circulating cell-free DNA is a potential candidate as such surrogate marker. Increased levels were found in murine and human post-stroke blood samples from the acute phase. In particular, cell-free double-strand DNA (dsDNA) binds to the innate immune sensor ”Absent In Melanoma 2” (AIM2)¹¹ in monocytes, which is inducing the formation of a high-molecular weight complex, the inflammasome. This leads to the cleavage-based activation of the inflammasome key protein caspase-1 and the vast secretion of IL-1b¹². This promotes a general state of systemic immune activation, which is represented by increased levels of IL-6, TNF-alpha and CCL2 amongst others. The knowledge of this mechanism and its clinical relevance also provides a multitude of therapeutical possibilities. A promising target is the blockage or degradation of the immunogenic dsDNA. Human recombinant desoxyribonuclease (hrDNase), approved as efficient treatment against dsDNA in cystic fibrosis, provides promising outcomes by degrading high concentrations of dsDNA after experimental stroke ameliorating systemic immune activation⁹. One major aspect remains unclear, which is the origin of the cell-free dsDNA in blood circulation after stroke: so far it is believed that the main DNA/DAMPs source is the necrotic neural tissue after stroke³. However, it is also plausible that early-activated neutrophils amplify the immune alterations by additional NET DNA secretion¹³. However, the mediator(s) for activating of this immune cell population and peripheral release of NETs has not yet been investigated in the context of systemic post-stroke immune alterations.

In AIM1 of this project we plan to study, if hyperacute neutrophil activation is caused by the alarmin ATP. Therefore, we use an established primary murine neutrophil in vitro assay. Neutrophils are stimulated with serum from mice which received a stroke or sham surgery. Afterwards, NETosis levels are investigated by quantifying citrullinated Histone3+ Sytox green (DNA)+ neutrophils. For a better understanding of the role of ATP in neutrophil activation, we plan to (1.) degradate DNA in the serum by using apyrase and analyze the NETosis levels. Moreover, we plan to (2.) antagonize the ATP binding to the neutrophils’ purinergic receptor P2X7R and analyze the NETosis status.

In AIM2 we plan to analyze NETosis inhibition in vivo with a PAD4 inhibitor or a purinergic receptor P2X7 inhibitor with multi-photon microscopy. In vivo labeling of neutrophils (fluorescent-labeled anti-Ly6G) and NETosis (Sytox green) are utilized to image intravascular activation of neutrophils in the hyperacute phase (2, 4 and 6 h) after stroke.