Myeloid-Biased HSC Require Semaphorin4a from the Bone Marrow Niche for Self-Renewal Under Stress and Life-Long Persistence

A subset of hematopoietic stem cells with inherent myeloid and platelet bias (myHSC) is positioned at the top of hematopoietic hierarchy and considered the most primitive. Notably, lower proliferative output of myHSC correlates with a greater capacity for self-renewal indicating that quiescence is essential for their function. Hence, excessive myHSC expansion following inflammatory challenge is likely to put them at a higher risk of proliferation-induced damage. Given that inflammatory stress is unavoidable throughout live, we hypothesized that myHSC may uniquely depend on quiescence-inducing signals for their protection and long-term persistence. However, the nature of these signals remains largely unexplored.

We have previously performed proximity-based analysis of the bone marrow niche to identify novel regulators of HSC quiescence (Silberstein et al, Cell Stem Cell 2016). Briefly, we defined the transcriptional profiles of osteolineage cells which were located in closer proximity to a transplanted HSC (proximal cells), and designated secreted factors with higher expression level in proximal cells as putative regulators of HSC quiescence. For the current study, we selected Semaphorin4a (Sema4a) - a known regulator of neural development, angiogenesis and immune response with no previously documented role in hematopoiesis - and examined its impact on myHSC function during inflammatory stress.

We found that Sema4a was expressed in the niche cells (endothelium and osteoprogenitors) in mice and humans. Recombinant Sema4a reduced proliferation of mouse and human hematopoietic stem and progenitor cells ex vivo. Baseline analysis of young Sema4aKO mice revealed mild anemia, thrombocytosis, myeloid bias and a slight reduction in the proportion of HSC in the G0 phase suggesting that Sema4a regulates HSC quiescence and differentiation in vivo. Upon Poly(I:C) injection, Sema4aKO myHSC (Lin ^-Kit ⁺Sca ⁺CD48 ^-CD34 ^-CD150 ^high) displayed markedly increased cycling and upregulation of alpha-interferon and JAK-STAT signaling while "balanced" HSC (Lin ^-Kit ⁺Sca ⁺CD48 ^-CD34 ^-CD150 ^low) were unaffected. Similar exaggerated proliferative response in Sema4aKO myHSC was observed upon injection with IL-1β.

Next, we investigated the long-term impact of inflammation-induced loss of myHSC quiescence. Aged Sema4aKO mice developed anemia, thrombocytosis, neutrophilia. Most significantly, we observed a two-fold expansion of phenotypic myHSC (but not balanced HSC) which displayed proliferative senescence, increased cellular stress and premature differentiation by scRNA-Seq, and a complete loss of reconstitution upon transplantation. In contrast, young Sema4aKO HSC showed a higher level of post-transplant chimerism consistent with their prior "pre-activated" state. Thus, loss of myHSC quiescence leads to increased sensitivity to inflammatory stressors and enhanced myHSC response but eventual collapse of regenerative function.

In order to determine if the microenvironment served as a critical source of Sema4a for myHSC, WT myHSC were transplanted into lethally irradiated WT and Sema4aKO hosts. Strikingly, the majority of Sema4aKO recipients died while all WT recipients survived. Intra-vital imaging at 24 hours revealed a greater number of cells and clusters in Sema4aKO recipients suggesting that excessive early myHSC proliferation led to impaired self-renewal and engraftment failure. Finally, we found that Plexin D1 acts as a functional receptor for Sema4a on myHSC, since Plexin D1-deficient myHSC recapitulated the post-transplant phenotype of young Sema4aKO myHSC described above.

Taken together, our data demonstrate that under the conditions of increased myeloid demand, protection from proliferative stress is critical for preserving myHSC function, and highlight a critical but previously unrecognized role for Sema4a-PlxnD1 axis in this process. Our study suggests that therapeutic augmentation of myHSC quiescence may alleviate the negative impact in inflammatory signaling, serve to improve marrow function in inflammatory diseases, and prevent development of myeloid malignancy.