WNK1-dependent macropinocytosis mediates emergency myelopoiesis

PROJECT SUMMARY/ABSTRACT Hematopoiesis ensures the continuous formation and turnover of blood cells during homeostasis, which shifts drastically towards production of inflammatory innate immune cells upon infection. At steady state, myeloid progenitors express a default signaling pathway essential for monocyte/macrophage production and require extrinsic signaling and transcription factors to direct lineage commitment towards other myeloid cell fates. During infection, coordination of cytokines and growth factors shifts myelopoiesis dramatically towards neutrophilia, a process known as emergency granulopoiesis. For instance, neutrophil production during systemic infection is boosted by over 10-fold, increasing cell numbers to over 1012 in a single day. Therefore, the rapid response to lineage-specific cytokines and growth factors to drastically increase granulocyte output is essential for pathogen clearance and host survival. Inappropriate lineage fidelity is exemplified by aberrant responses to infection observed in systemic inflammation and septic shock, however, the cellular machinery necessary to shift lineage commitment to emergency neutropoiesis remain poorly understood. Recently, we made the surprising discovery that the chloride-sensing kinase with-no-lysine 1 (WNK1) is indispensable for myeloid progenitor fate, with myeloid-specific deletion of Wnk1 incurring dramatic loss of tissue-resident macrophages, disrupted organ development, systemic neutrophilia, and mortality by 4 weeks old. Mechanistically, the cognate CSF1R cytokine, macrophage-colony stimulating factor (M-CSF), triggers macropinocytosis in myeloid progenitors, which in turn induces phosphorylation and activation of WNK1. Absence of WNK1 or macropinocytosis inhibition drove myeloid progenitor differentiation into granulocytes in vitro and in vivo. The goal of this proposal is to mechanistically dissect the WNK1-dependent pathway initiated by M-CSF-induced macropinocytosis and to investigate how this pathway is systematically shifted during emergency granulopoiesis. During the mentored phase of this application, I will master new techniques such as subcellular fractionation and macropinosome isolation. I will study how to analyze, integrate, and communicate complex functional genomics and metabolomics data. Together with colleagues, we will develop new tools to assess the importance of macropinocytosis during hematopoiesis in vivo, allowing us to understand how the contents of macropinosomes inform myeloid lineage commitment. With the guidance of my mentoring committee, I will strengthen my scientific and professional skillsets in preparation for the independent phase. During the independent phase, I will explore targeting the WNK1 pathway during sepsis using small molecules. Further, I will combine my expertise in in vivo sepsis models, emergency granulopoiesis, and neutrophil biology together with tools developed during the mentored phase to understand how macropinocytosis influences the innate immune response. The proposed project will allow me to successfully establish an independent academic research program.