Steady production of red and white blood cells from hematopoietic stem/precursor cells is required for normal homeostasis and immune system functions. Increased hematopoiesis is necessary in numerous pathologic settings, including following hemorrhage, microbial infections, and marrow ablation as part of the treatment for some leukemias before bone marrow transplantation [1
]. NF-κB signaling is activated in a variety of cell types in many of these settings and is required for immune cell formation and function [19
]. Thus, it is important to fully understand the mechanisms that regulate HSPCs in normal and disease states and the role of NF-κB in these states in particular. Previous studies have reported NF-κB canonical pathway involvement in HSPC functions, but it has been difficult to study this definitively because RelA−/− mice die during embryogenesis [22
], making it impossible to generate mice deficient in both RelA and p50, the major signaling proteins in the canonical pathway. Here, we generated RelB/NF-κB2 dKO mice to examine the role of non-canonical NF-κB signaling in the regulation of HSPC functions and have identified intrinsic roles for RelB and NF-κB2 in HSPCs and new and important extrinsic roles for them in cells in the marrow microenvironment.
RelB/NF-κB2 dKO mice have an overall ~2-fold increase in KLS marrow progenitor cells due to increased proliferation and decreased apoptosis, with only a slight, but significant decrease in LT-HSCs. This suggests an intrinsic role for the non-canonical pathway to maintain long-term stem cells or limit their differentiation or an extrinsic role or both. Importantly, however, dKO HSPCs were unable to functionally reconstitute the marrow of lethally irradiated WT mice in standard competitive, non-competitive, and serial transplantation experiments, and were out-competed by control HSPCs in chimeric mice. Thus, RelB/NF-κB2 intrinsically and positively regulate long-term stem cell self-renewal. Unlike the canonical pathway, which is activated quickly following ligand/receptor interaction, the non-canonical pathway is activated after several hours and following RelA/p50 induction of NF-κB p100 expression [19
]. Further study will be required to determine the factors that stimulate non-canonical signaling in HSPCs and the genes that are activated in them by RelB/NF-κB2.
We also found that RelB/NF-κB2 regulate hematopoiesis extrinsically through effects on the bone marrow microenvironment. An important role for osteoblasts in the marrow microenvironment to maintain hematopoiesis was recognized several years ago [10
], and since then roles for endothelial cells in hematopoiesis have been identified in a vascular niche [5
]. More recently, nestin-expressing bone marrow mesenchymal stem cells have been identified as another type of regulatory niche cell [7
]. We found that the frequency of bone marrow stromal cells, including that of immature stromal progenitor cells, is decreased in the dKO mice, indicating that the non-canonical pathway also intrinsically and positively regulates the bone marrow stromal population. In addition, dKO bone-lining cells displayed enhanced osteoblast differentiation, and dKO mice have increased bone volume (to be described in another publication), suggesting that this osteoblastic niche may have enhanced functions to compensate for the reduced number or activity of the stromal niche. However, our data showed that these bone-lining cells were unable to support hematopoietic expansion and differentiation as efficiently as controls. Molecularly, the dKO bone-lining cells express less Cxcl12, Thpo, osteopontin, and SCF (c-Kit ligand) than control cells. Thpo and Cxcl12, along with their receptors MPL and CXCR4, play essential roles in maintaining quiescence of the HSPC pool [37
]. Osteopontin maintains HSPC functions and keeps them attached to and in the osteoblastic niche [40
], and SCF stimulates HSPC proliferation [6
]. Thus, the decreased expression of these key players by dKO bone-lining cells could leave HSPCs in a state where they are freer to proliferate and differentiate because they have looser than normal contact to their niches, although these dKO bone-lining cells themselves do not support HSPC expansion. These data suggest that non-canonical signaling functions in the osteoblastic niche primarily to maintain HSPC-niche interactions and thus limit their differentiation into myeloid cells, which are increased in the dKO mice. These findings could also explain the results of transplantation of WT bone marrow cells into the dKO microenvironment: the WT HSPCs attain a relatively active status because they have less contact with the RelB/NF-κB2-null osteoblastic niche and they can expand because of the increased expression of other stimulatory factors (see below). Thus, non-canonical signaling appears to have an important role in bone marrow homeostasis coordinating the functions of both the stromal and osteoblastic niches to keep HSPCs quiescent. A key question needing further exploration is whether non-canonical signaling regulates the expression of these niche-interacting molecules by direct or indirect mechanisms.
Our data that wild-type donor hematopoietic cells proliferate faster in the dKO microenvironment and maintain their functional self-renewal capability when transplanted back to a control microenvironment, suggest that factors other than the less supportive HSPC function of dKO osteoblastic bone-lining cells are responsible for the HSPC expansion. To this end, we found that the expression levels of inflammatory cytokines capable of stimulating HSPC expansion and myeloid proliferation, including IL-6, G-CSF, and GM-CSF, were significantly increased in dKO bone marrow cells. Our in vitro culture experiments with dKO serum support this conclusion. Further support for an instructive role for these cytokines from the microenvironment comes from the results of 5-FU treatment and transplantation of WT bone marrow cells into the dKO microenvironment. For example, when either dKO bone marrow cells or wild-type donor cells were exposed to the dKO microenvironment, they became more myeloid and had less B lymphoid differentiation. In addition, after dKO bone marrow cells engrafted successfully in wild-type recipients, or wild-type donor cells “educated” in the dKO microenvironment were transplanted back into wild-type recipients, lineage commitment reverted almost completely to normal. Furthermore, although dKO KLS cells proliferated faster, they were phenotypically resistant to 5-FU treatment, and when WT cells were in the dKO microenvironment, the WT cells became 5-FU-resistant. However, we realize that after transplantation of WT bone marrow cells into dKO mice, and even when dKO recipients were stable and had higher chimerism, the donor cells were still skewed to the myeloid lineage and proliferated faster. One explanation is that even after 16 weeks, an inflammatory cell infiltrate could still be identified in the organs we examined, including lung and liver, although the severity was greatly reduced compared to dKO mice without transplantation (Supporting information Fig. 10
). These data suggest that local and systemic inflammatory cytokines are responsible for the enhanced hematopoiesis.
The increased myelopoiesis in our dKO mice is also seen in IκBα−/− mice, which was attributed to a hepatocyte/bone marrow stroma-mediated effect on myeloid lineage cells [43
]. This provides further support for the possibility that inflammatory cytokines released from other organs in our dKO mice may also be responsible for the enhanced hematopoiesis. However, we cannot exclude the possibility of the existence of other key as yet unidentified cytokines. Interestingly, earlier studies have reported increased absolute and relative HSPC numbers in chronic infection [44
]. Currently, whether and how infection affects non-canonical and canonical signaling in HSPCs and their niches is unknown. Nevertheless, our data at least partially explain the myeloid proliferative phenotype and the increased HSPCs in the dKO bone marrow microenvironment.