Transcriptional regulation of IL-15 expression during hematopoiesis.

Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8⁺ and CD8⁻). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineage⁻Sca-1⁺c-Kit⁺) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15⁺CD8⁺ and IL-15(⁻/low)CD8⁻ DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

A Phe377del mutation in ANK leads to impaired osteoblastogenesis and osteoclastogenesis in a mouse model for craniometaphyseal dysplasia (CMD).

Craniometaphyseal dysplasia (CMD) is a rare genetic disorder with hyperostosis of craniofacial bones and widened metaphyses in long bones. Patients often suffer from neurological symptoms due to obstruction of cranial foramina. No proven treatment is available and the pathophysiology is largely unknown. A Phe377 (TTC(1130-1132)) deletion in exon 9 of the pyrophosphate (PPi) transporter ANK leads to CMD-like features in an Ank(KI/KI) mouse model. Here, we investigated the effects of CMD-mutant ANK on mineralization and bone mass at a cellular level. Ank(KI/KI) osteoblast cultures showed decreased mineral deposition. Expression of bone mineralization regulating genes Mmp13, Ocn, Osx and Phex was reduced in Ank(KI/KI) osteoblasts, while the Fgf23 mRNA level was highly elevated in Ank(KI/KI) calvarial and femoral bones. Since ANK is a known PPi transporter, we examined other regulators of Pi/PPi homeostasis Enpp1 and Tnap. Significantly increased ENPP1 activity may compensate for dysfunctional mutant ANK leading to comparable extracellular PPi levels in Ank(+/+) osteoblasts. Similar to Ank(KI/KI) bone marrow-derived macrophage cultures, peripheral blood cultures from CMD patients exhibited reduced osteoclastogenesis. Cell-autonomous effects in Ank(KI/KI) osteoclasts resulted in disrupted actin ring formation and cell fusion. In addition, Ank(KI/KI) osteoblasts failed to adequately support osteoclastogenesis. Increased bone mass could partially be rescued by bone marrow transplants supporting our hypothesis that reduced osteoclastogenesis contributes at least in part to hyperostosis. We conclude that the Phe377del mutation in ANK causes impaired osteoblastogenesis and osteoclastogenesis resulting in hypomineralization and a high bone mass phenotype.

Hematopoietic and stromal DMP1-Cre labeled cells form a unique niche in the bone marrow.

Skeletogenesis and hematopoiesis are interdependent. Niches form between cells of both lineages where microenvironmental cues support specific lineage commitment. Because of the complex topography of bone marrow (BM), the identity and function of cells within specialized niches has not been fully elucidated. Dentin Matrix Protein 1 (DMP1)-Cre mice have been utilized in bone studies as mature osteoblasts and osteocytes express DMP1. DMP1 has been identified in CXCL12+ cells and an undefined CD45+ population. We crossed DMP1-Cre with Ai9 reporter mice and analyzed the tdTomato+ (tdT+) population in BM and secondary hematopoietic organs. CD45+tdT+ express myeloid markers including CD11b and are established early in ontogeny. CD45+tdT+ cells phagocytose, respond to LPS and are radioresistant. Depletion of macrophages caused a significant decrease in tdT+CD11b+ myeloid populations. A subset of CD45+tdT+ cells may be erythroid island macrophages (EIM) which are depleted after G-CSF treatment. tdT+CXCL12+ cells are in direct contact with F4/80 macrophages, express RANKL and form a niche with B220+ B cells. A population of resident cells within the thymus are tdT+ and express myeloid markers and RANKL. In conclusion, in addition to targeting osteoblast/osteocytes, DMP1-Cre labels unique cell populations of macrophage and stromal cells within BM and thymus niches and expresses key microenvironmental factors.

Splenomegaly, myeloid lineage expansion and increased osteoclastogenesis in osteogenesis imperfecta murine.

Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b+) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters.

Sex and genetic factors determine osteoblastic differentiation potential of murine bone marrow stromal cells.

Sex and genetic factors determine skeletal mass, and we tested whether bone histomorphometric parameters were sexually dimorphic in femurs from 1 to 6 month old C57BL/6 mice. Trabecular bone volume declined more rapidly in female mice than in male littermates because of enhanced bone resorption. Although bone formation was not different between sexes, female mice exhibited a higher number of osteoblasts than male littermates, suggesting that osteoblasts from female mice may have a reduced ability to form bone. To determine the impact of sex on osteoblastogenesis, we investigated the potential for osteoblastic differentiation of bone marrow stromal cells from C57BL/6, Friend leukemia virus-B (FVB), C3H/HeJ and BALB/c mice of both sexes. Bone marrow stromal cells from female FVB, C57BL/6 and C3H/HeJ mice exhibited lower Alpl and Osteocalcin expression and alkaline phosphatase activity, and formed fewer mineralized nodules than cells from male littermates. Proliferative capacity was greater in cells from male than female C57BL/6, but not FVB, mice. Sorting of bone marrow stromal cells from mice expressing an α-Smooth muscle actin-green fluorescent protein transgene, revealed a higher yield of mesenchymal stem cells in cultures from male mice than in those from female littermates. Sex had a modest impact on osteoblastic differentiation of mesenchymal stem cells. To determine the influence of sex and genetic factors on osteoblast function, calvarial osteoblasts were harvested from C57BL/6, FVB, C3H/HeJ and BALB/c mice. Alpl expression and activity were lower in osteoblasts from C57BL/6 and C3H/HeJ, but not FVB or BALB/c, female mice than in cells from littermates. Sex had no effect on osteoclastogenesis of bone marrow cultures of C57BL/6 mice, but osteoblasts from female mice exhibited higher Rankl and lower Opg expression than cells from male littermates. In conclusion, osteoblastogenesis is sexually dimorphic and influenced by genetic factors.

Deletion of CD74, a putative MIF receptor, in mice enhances osteoclastogenesis and decreases bone mass.

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF-regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild-type (WT) and CD74-deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8-week-old mice was measured by micro-computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast-like cells (OCLs) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate-resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL-induced-signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL-induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c-Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL-induced activator protein 1 (AP-1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis.

Identification, characterization, and isolation of a common progenitor for osteoclasts, macrophages, and dendritic cells from murine bone marrow and periphery.

Osteoclasts are specialized bone-resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype B220(-) CD3(-) CD11b(-/low) CD115(+) and either CD117(hi), CD117(intermediate), or CD117(low). We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single-cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages, and antigen-presenting dendritic cells in vitro with efficiencies of more than 90%, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages, and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined.

Chemotactic and immunoregulatory properties of bone cells are modulated by endotoxin-stimulated lymphocytes.

In our study, we explored the bidirectional communication via soluble factors between bone cells and endotoxin-stimulated splenic lymphocytes in an in vitro coculture model that mimics the inflammatory environment. Both the ability of lymphocytes to affect differentiation and immune properties of bone cells, osteoblasts (OBL) and osteoclasts (OCL), and of bone cells to modulate cytokine and activation profile of endotoxin-stimulated lymphocytes were tested. LPS-pulsed lymphocytes enhanced OCL but inhibited OBL differentiation and increased the RANKL/OPG ratio, and, at the same time, upregulated chemotactic properties of bone cells, specifically CCL2, CCL5, and CXCL10 in OCL and CCL5 and CXCL13 in OBL. In parallel, bone cells had immunosuppressive effects by downregulating the lymphocyte expression of interleukin (IL)-1, IL-6, TNF-α and co-stimulatory molecules. OCL stimulated the production of osteoclastogenic cytokine RANKL in T lymphocytes. The anti-inflammatory effect, especially of OBL, suggests a possible compensatory mechanism to limit the inflammatory reaction during infection.

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow.

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1-34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115(+) CD117(high) and TN/CD115(+) CD117(int) cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115(+) CD117(low) cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF-κB ligand (RANKL)- and macrophage colony-stimulating factor (M-CSF)-stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115(+) cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin(-) CD117(+) Sca-1(+) ) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M-CSF and RANKL.

Essential role of the RNA-binding protein HuR in progenitor cell survival in mice.

The RNA-binding protein HuR (also known as ELAV1) binds to the 3'-untranslated region of mRNAs and regulates transcript stability and translation. However, the in vivo functions of HuR are not well understood. Here, we report that murine HuR is essential for life; postnatal global deletion of Elavl1 induced atrophy of hematopoietic organs, extensive loss of intestinal villi, obstructive enterocolitis, and lethality within 10 days. Upon Elavl1 deletion, progenitor cells in the BM, thymus, and intestine underwent apoptosis, whereas quiescent stem cells and differentiated cells were unaffected. The survival defect of hematopoietic progenitor cells was cell intrinsic, as transplant of Elavl1-/- BM led to compromised hematopoietic reconstitution but did not cause lethality. Expression of p53 and its downstream effectors critical for cell death were induced in progenitor cells as HuR levels declined. In mouse embryonic fibroblasts, HuR bound to and stabilized the mRNA for Mdm2, a critical negative regulator of p53. Furthermore, cell survival was restored by expression of Mdm2 in Elavl1-/- cells, suggesting that HuR keeps p53 levels in check in progenitor cells and thereby promotes cell survival. This regulation of cell stress response by HuR in progenitor cells, which we believe to be novel, could potentially be exploited in cytotoxic anticancer therapies as well as stem cell transplant therapy.

Regulation of hematopoietic niches by sympathetic innervation.

Once hematopoiesis is established in the bone marrow, a continuous egress of hematopoietic stem cells (HSCs) to the periphery occurs at a low frequency. It has been proposed that this phenomenon is part of a regenerative homeostatic mechanism that ensures the maintenance of hematopoiesis through the life of the individual. The administration of certain cytotoxic drugs or cytokines can enhance the mobilization of hematopoietic progenitors to the periphery. During the past 15 years, granulocyte-colony stimulating factor (G-CSF) has been used as a standard cytokine for mobilization protocols in experimental models and in humans. Despite extensive efforts by multiple groups, a definitive mechanism explaining its role in mobilization has not been provided. In a recent paper, Katayama et al., through a series of clever associations supported by well-defined experimental systems, proposed that signals through the sympathetic nervous system modify the activity of the hematopoietic niche, acting as regulators of the mobilization of hematopoietic progenitors. This surprising finding adds a new level of complexity to the cellular milieu responsible for generation and maintenance of the hematopoietic niche.

Skeletal development, bone remodeling, and hematopoiesis.

In adult mammals, the bone marrow microenvironment is defined by close interactions between cells derived from mesenchymal progenitors and cells derived from hematopoietic progenitors. The influence that one population of cells has over the other has been a matter of intense study since it was established that hematopoietic stem cells (HSCs) require support of stromal elements to engraft, self-renew, and progress towards lineage commitment. Within the stromal components, cells of the osteoblastic lineage have the ability to interact with HSCs, and it has been proposed that they could be one of the main cell types responsible for the generation and maintenance of hematopoietic niches. Possible molecular mechanisms involved in the interaction between osteoblastic and hematopoietic cells have been described. However, understanding the relative importance of each one of them, their production by defined cells, and their kinetics of appearance have been limited by the lack of in vivo models allowing the physical and/or temporal dissection of the components of the osteoblastic lineage. Here, we provide a summary of the evidence that have established the importance of osteoblasts in hematopoiesis, and we propose new experimental strategies that could help to define the nature of these interactions.