Hematopoietic stem and progenitor cells (HSPCs) spend their lives making mature blood cells. In the process, HSCs interact with a variety of cells in the bone marrow microenvironment, including macrophages. These macrophages mediate a range of processes by releasing cytokines and chemokines and patrolling to remove stressed, dead or aged cells. In this way, macrophages contribute to the maintenance of tissue homeostasis.
During hematopoiesis, macrophages ensure the quality of normal HSCs and determine the number of HSC clones that participate in adult hematopoiesis. Macrophages then either completely engulf the stem cells (called "dooming") or capture part of the stem cells' cellular material (called "grooming"). Groomed stem cells continue to divide, whereas the doomed HSPC clone is eliminated.
Past studies have shown that the interaction between HSPCs and macrophages is mediated by the "eat me" signal calreticulin (Calr) on the surface of HSPCs. Hematopoietic stem cells with high levels of reactive oxygen species (ROS) have elevated levels of surface Calr, which triggers macrophages to engulf and kill stem cells containing a large number of stress-activated proteins. However, the specific molecular clues that regulate "engulfing" and "grooming" behaviors remain unknown.
In a new study, in order to study the clues that mediate engulfing and grooming behaviors, researchers from Boston Children's Hospital and Harvard University and other research institutions screened 1,200 bioactive small molecules in human cells and found that 93 compounds could significantly increase surface CALR in a dose-dependent manner. Among these compounds, 22 also promoted the interaction between macrophages and stem cells in zebrafish.
Compounds that increase CALR in a ROS-dependent manner exhibited higher dooming ratios. In contrast, zebrafish treated with ROS-independent compounds had a higher rate of grooming events despite increased CALR and increased macrophage-stem cell interactions. To investigate the signals involved in the interaction under ROS-independent conditions, the researchers performed a genome-wide screen in human cells. They identified Toll-like receptor 3 (TLR3) as an inducer of CALR in the "don't eat me" context.
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Next, the researchers found that TLR3 expression was required for the expression of the "don't eat me" signal beta-2-microglobulin (B2M). Antibody staining and studies with a b2m knockout zebrafish strain showed that b2m on the stem cell surface is required to prevent macrophage phagocytosis. To determine whether the increased phagocytosis observed in b2m mutants affects clonal dominance, they generated a mosaic deletion using the zebrafish color barcoding system (TWISTR) as a lineage tracker. Mosaic deletion of b2m reduced the number of myeloid clones but increased clonal dominance. The dominant clones in these zebrafish were wild-type b2m, which were resistant to macrophage phagocytosis. TLR3 signaling is activated by double-stranded RNA. Double-stranded RNA is sometimes found in cells due to the expression of transcripts associated with repetitive elements (REs), including long terminal repeats (LTRs) and endogenous retroviruses.
Expression of repetitive elements in HSPCs positively correlated with increased levels of B2m. Furthermore, overexpression of ltr4 in zebrafish resulted in higher HSPC proliferation, indicating reduced macrophage phagocytosis. Disruption of the TLR3-B2M pathway by interfering with the expression of TLR3, IRF3, or B2M reduced the number of zebrafish hemocyte clones. Use of the DNA methyltransferase inhibitor CM272 to promote upregulation of repetitive elements expanded the proliferation of established HSPC clones in zebrafish embryos.
Figure 1. ROS and TLR3 signaling mediate "eat me" and "don't eat me" signals that determine macrophage selection of stem cell clones. (Pessoa Rodrigues C, et al., 2024)
Taken together, these findings support a model in which endogenous TLR3 ligands lead to the expression of B2M, which blocks macrophage-induced phagocytosis and determines the clonality of hematopoietic stem cells by monitoring stem cell quality with macrophages. The balance of Calr and B2M controls the number of long-lived hematopoietic stem cell clones that contribute to the adult blood system through macrophage-mediated quality assurance. This reveals an intricate interplay between HSPC clonal expansion and clearance by immune regulatory cells. The researchers propose that this protective mechanism may also play a role in adulthood in response to environmental stress, such as infection or clonal stem cell dysregulation. Manipulating the levels of "don't eat me" and "eat me" signals may have important therapeutic implications for immunotherapies that use macrophages to selectively eliminate mutant stem cell clones.
Reference
Pessoa Rodrigues C, et al. Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells. Science, 2024, 385(6714): eadn1629.