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.
