Blood platelets, essential to stop bleeding, are produced by large cells knowns as megakaryocytes, which develop from hematopoietic stem cells in the bone marrow. Recent advancements, especially in single-cell RNA sequencing (scRNA-seq), have revealed distinct subsets of megakaryocytes, including an immune subset. The exact process and location of how platelets are formed by megakaryocytes are not well understood. However, evidence indicates that the microvasculature of the lung may be crucial in the final step of platelet generation, assisting in the fragmentation of megakaryocyte protrusions into individual platelets. Inflammation has been shown to affect platelets; however the underlying mechanisms remain mostly unknown.

P04 aims to better understand how inflammation influences megakaryocyte differentiation, maturation and platelet biogenesis by focusing on two main objectives: 1) to assess the impact of systemic inflammatory disease settings on the megakaryocyte transcriptome and the generated platelets, and 2) to investigate the influence of thrombo-inflammation on platelet generation in the pulmonary microvasculature. To this end, we will induce sepsis or endotoxaemia in mice, and subject megakaryocytes to scRNA-seq. Transcriptional changes will be identified in MKs and correlated to protein expression in platelets by immunoblotting. In parallel, the process of platelet formation in the lung will be examined using a megakaryocyte infusion model, with a focus on understanding how inflammation influences this mechanism.

ECR.P04.05 will focus on isolating and enriching megakaryocytes from mice undergoing systemic inflammation, and will subject these cells to scRNA-seq to analyze their gene expression profiles. Data analysis will be performed in the programming language R using the Seurat package. Identified top up/down-regulated genes will be verified in megakaryocytes using bone marrow cryosections and immunostainings and in platelets using an automated capillary-based immunoblotting platform or flow cytometry. Following the identification of specific up-regulated proteins amenable to in vivo manipulation, the PhD candidate will test whether these could be used as targets of experimental intervention. In addition, the PhD candidate will perform megakaryocyte infusion experiments to follow their fate in vivo, mainly focusing on the lung. These studies will involve advanced mouse models, antibody-based labelling protocols and cutting-edge intravital microscopy. The impact of inflammation on platelet generation in the lung will also be explored.