Oxidative stress in mutated neutrophils fosters thrombopoiesis in Jak2VF clonal hematopoiesis driven atherosclerosis

J. Pircher (München)¹, Q. Ul Ain (München)¹, Z. Zhang (München)¹, L. Wasner (München)¹, M. A. Hajebrahimi (München)¹, A. Tall (New York)², N. Wang (New York)², D. Stegner (Würzburg)³, T. Petzold (Berlin)⁴, S. Massberg (München)¹, C. Schulz (Mannheim)^5
1LMU Klinikum der Universität München Medizinische Klinik und Poliklinik I München, Deutschland; ²Columbia University New York, USA; ³Universitätsklinikum Würzburg Inst. für Exp. Biomedizin, Lehrstuhl f. Exp. Biomed. II Würzburg, Deutschland; ⁴Deutsches Herzzentrum der Charite (DHZC) Klinik für Kardiologie, Angiologie und Intensivmedizin Berlin, Deutschland; ⁵Universitätsmedizin Mannheim der Universität Heidelberg MANNHEIM INSTITUTE for INNATE IMMUNOSCIENCE Mannheim, Deutschland

We recently described a prothrombotic phenotype in Jak2^VF-driven clonal hematopoiesis (CH) which was in part explained by increased production of hyper-reactive young platelets. Further, oxidative stress in neutrophils plays a role in Jak2^VF associated MPN. We previously showed that neutrophil-megakaryocyte crosstalk drives prothrombotic thrombopoiesis in a ROS- and CXCR4-dependent manner. Here, in a mouse model of Jak2^VF CH we observed upregulated NOX-subunits in myeloid cells, which was more pronounced in atherosclerosis. Consequently, neutrophils in Jak2^VF-CH showed increased baseline levels of intracellular ROS. Further, elevated neutrophil counts were associated with increased reticulated platelet counts indicating increased production. Analysis of bone marrow thrombopoiesis by multiphoton imaging next to accelerated proplatelet release in Jak2^VF CH revealed more megakaryocyte-neutrophil-interactions around the proplatelet budding site, that we previously showed to boost release of reactive young platelets. Using fluorescent reporter-mice we analyzed behavior of Jak2^VF-mutated or wildtype cells within the same individual. While Jak2^VF neutrophils preferentially interacted with megakaryocytes, Jak2^VF megakaryocytes showed similar proplatelet release kinetics as their wildtype counterparts. In line with this Jak2^VF neutrophils showed increased CXCR4-surface expression and PMA-induced ROS production as compared to wildtype cells. Eventually antioxidative treatment with N-acetyl-cysteine reduced proplatelet release resulting in decreased peripheral platelets. Together, we describe increased ROS production by mutated neutrophils and megakaryocyte-interaction as additional mechanism contributing to prothrombotic thrombopoiesis in JAK2^VF CH.