Macrophages mediate hematopoietic stem cell trans-endocytosis to coordinate regenerative stress and aging

Abstract The hematopoietic stem cell (HSC) niche is a specialized microenvironment in the bone marrow (BM) that controls HSC activity to ensure blood homeostasis. Macrophages have been suggested to be a critical niche component that regulate HSC retention and fate decisions (Chow A, J Exp Med 2011; Zhang D, Cell Stem Cell 2022). We recently showed that HSCs acquired surface proteins from macrophages through a membrane transfer process termed trogocytosis, which conferred HSC residency in the BM (Gao X, Science 2024). However, the underlying mechanisms and relevance of HSC trogocytosis in normal and stress conditions remain largely unclear.To investigate the role of macrophages in trogocytosis, we first established an approach to separate bona fide macrophages from the trogocytosis population in the BM. We found that the combination of CD169 and MerTK clearly separated F4/80+macrophages into three subpopulations, R1, R2 and R3. Notably, macrophage-specific gene expression and phagocytic activity were only enriched in R1, and F4/80+ HSCs with long-term reconstitution capacity were exclusively contained in R2, suggesting that R1 cells are bona fide macrophages, and R2 cells represent the trogocytosis population. We then compared these populations and found that CD169 showed higher protein levels on R2 but was only transcribed in R1 cells, indicating that this protein was actively transferred. We then analyzed CD169-/- mice and found that R2 cells and macrophage marker expressions on HSCs were completely abolished, suggesting that CD169 was essential in mediating the transfer.Next, we asked whether the trogocytosis phenomenon is commonly presented in hematopoietic tissues. Despite the abundant expression of CD169 on splenic and liver macrophages, R2 cells and macrophage marker expressions on HSCs were only observed in the BM. Interestingly, α-2,6-linked sialic acid, the ligand of CD169, was highly presented on HSCs in the BM, but reduced to low levels in the periphery, suggesting that the ligand levels may control where trogocytosis occurred. To understand how CD169 mediated the transfer, we analyzed the subcellular structures of HSCs by transmission electron microscopy, and observed abundant membrane fragments on the surfaces of HSCs, which was significantly reduced in CD169-/- mice, suggesting that membrane fragments were the primary form of transfer.Further, we investigated whether HSCs may uptake transferred materials by culturing BM cells in vitro. We observed a “self-grooming” phenomenon marked by rapid clearance of transferred proteins in culture, which was associated with increased uptake of pHrodo-dextran, an endocytic indicator. The self-grooming behavior was prevented by endocytosis inhibitors, and immunofluorescence analyses revealed enhanced co-localization with lysosomes and reduced volumes of CD169 in cultured HSCs, suggesting that transferred membrane was endocytosed and transported to lysosome for degradation. Interestingly, we found that lactate treatment completely inhibited the trans-endocytic activity of HSCs. Consistently, cultured HSCs exhibited significantly reduced levels of lysine lactylation, which was fully reversed by lactate treatment, indicating that this post-translational modification may be essential for the regulation of trans-endocytic activity by the microenvironment.Finally, we evaluated whether HSC trans-endocytosis may lead to nutrient influx and metabolic regulation. To this end, we assessed mTOR activity and mitochondrial parameters, and found that S6K and mTOR phosphorylation, mitochondrial mass and ROS production, but not membrane potential or oxygen consumption rate of HSCs, were reduced in CD169-/- mice, without altering the long-term reconstitution capacity. However, when CD169-/- mice were challenged with 5-fluorouracil to induce myeloablation, we observed significantly altered HSC response with dysregulated self-renewal and differentiation during regeneration. We further analyzed whether defects in trans-endocytosis may perturb HSC fate decisions during hematopoietic aging. Strikingly, aged CD169-/- mice exhibited dramatic expansion of HSCs (~87.5-fold change), and significant increases of long-term reconstitution following competitive BM and sorted HSC transplantations. Collectively, our data have uncovered a role for BM macrophages in mediating HSC trans-endocytosis, which plays an essential role in coordinating HSC fate decisions during regenerative stress and aging.