Radio-resistant recipient bone marrow (BM) macrophages (MACS) are necessary for hematopoietic stem cell (HSC) engraftment post transplantation

Abstract

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BM-Macs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-Lin-Kit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP+ HSC. Depletion of recipient CD169+ Macs abated engraftment of donor phenotypic HSC by 70% at 5 wk post-transplant and reduced BM reconstitution potential in competitive secondary transplants. In conclusion BM contains a myeloablation-resistant self-repopulating Mac subset that is necessary for efficient and/or sustained HSC BM engraftment following transplantation. Therefore interpretation of transplantation chimera experiments must take into account the persistence of this subset of host macrophages that support donor HSC engraftment.