Abstract 383: MLKL (Mixed Lineage Kinase Domain-like Protein) In The Splenic Microenvironment Restrains Hematopoietic Contributions To The Atherosclerotic Plaque

Abstract

The mixed lineage kinase domain-like protein (MLKL) was first discovered in 2012 as the executioner of necroptosis. In line with this, we showed that MLKL knockdown ( Mlkl KD) by administration of antisense oligonucleotides to atherosclerotic-prone Apoe -knockout ( Apoe -/-) mice did indeed decrease necroptosis and necrotic core in the atherosclerotic plaque. However, there were no changes in overall plaque area, suggesting additional roles of MLKL during atherosclerosis. During atherogenesis, the spleen allows for the expansion of hematopoietic stem and progenitor cells (HSPCs) and mature myeloid cells that drive atherogenesis. Accordingly, we observed splenomegaly with an expansion of splenic HSPCs and mature myeloid cells upon Mlkl KD. However, bone marrow transplantation studies revealed that MLKL did not impact hematopoiesis through its expression in hematopoietic cells. Within hematopoietic reservoirs such as the spleen, HSPCs are regulated by their interaction with microenvironmental cell types, including endothelial cells. We thus hypothesized that MLKL preserves the microenvironment to repress hematopoiesis in the spleen during atherosclerosis. Flow cytometry and immunofluorescence staining during a time course of atherosclerosis revealed a progressive decrease of splenic endothelial cells that was correlated with a reduction of MLKL endogenously or by Mlkl KD, suggesting that splenic MLKL modulates the endothelial cells known to regulate hematopoiesis. RNA sequencing of these splenic endothelial cells revealed two major pathways that were perturbed upon Mlkl KD, including cell cycle and factors related to leukocyte differentiation. Adoptive transfer of CD45.1 HSPCs to CD45.2 Apoe -/- mice during Mlkl KD revealed an expansion of mature immune cells in the spleen as compared to control, together demonstrating the influence of MLKL on the splenic microenvironment and subsequently hematopoiesis. In conclusion, we here demonstrate a novel role for MLKL in regulating the balance of hematopoiesis, specifically through preservation of splenic endothelial cells that promote HSPC quiescence, and more generally highlight the contributions of the spleen and its impact on inflammatory pathways that potentiate atherogenesis.