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CK2-dependent cytoskeletal regulation and molecular signaling of Neutrophil Extracellular Trap (NET) formation

Periodic Reporting for period 2 - COAGULANT (CK2-dependent cytoskeletal regulation and molecular signaling of Neutrophil Extracellular Trap (NET) formation)

Reporting period: 2020-09-01 to 2021-08-31

Thrombo-inflammatory diseases such as myocardial infarction, ischemic stroke, and venous thromboembolism, are still a leading cause of morbidity and mortality in the European Union. In the last decade, and even more recently during the ongoing SARS-CoV-2 pandemic, there is a growing body of evidence showing a pivotal role of polymorphonuclear neutrophils and especially Neutrophil Extracellular Trap formation (NETosis) in the development and progression of thrombo-inflammation.
Beside its clinical implication, only rudimentary cellular processes and the time course of NETosis could be just recently unraveled, but are still not understood in detail. However, the identification of intracellular molecular pathways and signaling proteins in NETosis is of crucial importance for the development of possible new treatment regimens. The consequent objective of the MSCA was the investigation of molecular signaling pathways which are important for NETosis and which subsequently affect or predict thrombosis in human patients.
In this regard, the NLRP3 inflammasome assembly could be defined as pivotal process during NETosis and subsequent venous thrombosis. Moreover, it could be demonstrated that the PAD4 enzyme, which usually regulates NET-associated venous thromboembolism, also affects NLRP3 inflammasome assembly by regulating the protein levels of the most important inflammasome components NLRP3 and ASC. Finally, the obtained findings link the canonical inflammasome signaling and NETosis. This leads to the conclusion that diseases, which are associated with increased PAD4 expression in neutrophils and elevated susceptibility to NETosis, could possibly be linked to elevated NLRP3 inflammasome assembly in both neutrophils and macrophages. Therefore, the PAD-dependent regulation of NLRP3 protein levels could be an important mechanism in inflammasome-driven diseases and makes PADs promising new targets in the therapy of cardiovascular diseases and even opens many new avenues for new approaches in the prevention and treatment of inflammatory diseases.
The work performed as part of the MSCA unraveled the PAD4-dependent assembly of the NLRP3 inflammasome as important part of NETosis and subsequent venous thrombosis under sterile conditions and is published in a peer-reviewed and open-access journal (Münzer et al., Front. Immunol. 2021).
During the outgoing phase, neutrophils were isolated from NLRP3- and PAD4-deficient mice, the corresponding wild-type littermates, and from human blood samples. By employing in vitro NET assays and different immunofluorescence techniques, an effect of NLRP3 inflammasome assembly on NETosis was demonstrated. Moreover, using Western Blot and PCR approaches, a PAD4-dependent regulation of the inflammasome protein components NLRP3 and ASC was shown. To pinpoint this observation, bone marrow-derived macrophages were genetically engineered to overexpress the PAD4 enzyme, which confirmed the observation in primary neutrophils. Additionally, a newly established workflow of NETosis visualization was established to investigate the cellular effect of NLRP3 deficiency on NETosis, which revealed an effect of NLRP3 on nuclear envelope and plasma membrane breakage. As completion of the outgoing phase an in vivo model of NET-dependent deep vein thrombosis was performed in NLRP3-deficient and wild-type mice and confirmed a physiological relevance of the findings.
In the return phase, the above mentioned methods and knowledge were transferred to EKUT. Thereby, in particular the establishment of the stenosis-induced in vivo mouse model of deep vein thrombosis perfectly complete the existing methodology. Furthermore, the establishment of the high-resolution time-lapse imaging of living neutrophils was just recently started and will be extended to the imaging of myocardial cells and even platelets in near future. As proposed in the original DoA the sampling and collection of cardiovascular and thrombo-inflammatory patient cohorts started to identify new biomarkers and predictive factors of thrombo-occlusive disorders. So far, first experiments were started and preliminary results show a strong effect of NLRP3 on NETosis in the human system too. To obtain more solid data in human patient cohorts more work and investigations will be done in future, so that the datasets can be exploited in a translational approach together with the pharmaceutical industry.
In conclusion, in line with the MSCA, the NLRP3 inflammasome was identified as crucial part of NETosis with subsequent deep vein thrombosis in mice. Moreover, it could be demonstrated that the PAD4 enzyme regulates the protein levels of the most important inflammasome components NLRP3 and ASC, thus opening new avenues for possible new pharmaceutical interventions and strategies.
The underlying MSCA created several connecting points for further projects and investigations. The results and observations significantly contribute to the understanding of the cellular mechanism of NETosis and subsequent inflammatory processes such as thrombo-inflammatory disorders, which are affecting the life quality and well-being of European citizens tremendously.
In particular, the connection of the canonical inflammasome to NETosis may open new possibilities in the treatment of inflammatory diseases. On the one hand, the finding that PAD4 regulates inflammasome assembly and cell priming make PAD enzymes a promising pharmacological target in the therapy of thrombo-inflammatory and inflammasome-associated diseases. On the other hand, already existing inhibitors of NLRP3 such as MCC950 possibly are also effective in the reduction of the toxic effects of NETosis. These potential new fields of application of already used pharmacological compounds could help to minimize the enormous health care costs in the EU and improve the life quality and well-being of European citizens. However, the pharmacological exploitation of these possibilities needs further investigations. Thus a close collaboration with the pharmaceutical industry and translational bench-to-bedside approaches are intended in the near future to provide a fast, cheap and reliable treatment and prevention of thrombo-inflammatory disorders to the European citizens.
However, especially in regard to the still ongoing SARS-CoV-2 pandemic and its impact on the European economy and society, the knowledge of cellular and molecular pathways leading to NETosis may even be helpful to fight the devastating consequences of a SARS-CoV-2 infection, since NETosis and NET-associated extracellular factors significantly contribute to the clinical outcome of COVID-19 patients. Consequently, a plurality of clinical trials are performed investigating the effect of NET resolution, NETosis inhibition in general NET-associated inhibition of the IL-1 receptor on COVID-19-related immuno-thrombosis or to develop an easy accessible diagnostic test of COVID-19. In the efforts to develop NET-based therapeutic and diagnostic tools for COVID-19, the results from the underlying MSCA help to understand the role of NLRP3 in neutrophil function and thus give insights in a highly inflammatory aspect of NETosis.
Altogether, the investigations and results in line with the MSCA contribute to the understanding of the detailed cellular and molecular pathways of NETosis and has potential impact on a wide variety of inflammatory and cardiovascular diseases.
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