Abstract
The contributions of skeletal cells to the processes of B cell development in the bone marrow (BM) have not been completely described. The von-Hippel Lindau protein (VHL) plays a key role in cellular responses to hypoxia. Previous work showed that Dmp1-Cre;Vhl conditional knockout mice (VhlcKO), which deletes Vhl in subsets of mesenchymal stem cells, late osteoblasts and osteocytes, display dysregulated bone growth and reduction in B cells. Here, we investigated the mechanisms underlying the B cell defects using flow cytometry and high-resolution imaging. In the VhlcKO BM, B cell progenitors were increased in frequency and number, whereas Hardy Fractions B-F were decreased. VhlcKO Fractions B-C cells showed increased apoptosis and quiescence. Reciprocal BM chimeras confirmed a B cell-extrinsic source of the VhlcKO B cell defects. In support of this, VhlcKO BM supernatant contained reduced CXCL12 and elevated EPO levels. Intravital and ex vivo imaging revealed VhlcKO BM blood vessels with increased diameter, volume, and a diminished blood-BM barrier. Staining of VhlcKO B cells with an intracellular hypoxic marker indicated the natural existence of distinct B cell microenvironments that differ in local oxygen tensions and that the B cell developmental defects in VhlcKO BM are not initiated by hypoxia. Our studies identify novel mechanisms linking altered bone homeostasis with drastic BM microenvironmental changes that dysregulate B cell development.
Highlights
The mechanisms by which changes in bone homeostasis affect immune development in the bone marrow (BM) are not fully understood [1–4]
Similar to previous reports [21], we found that long bones in B6 Vhl conditional knockout mice (VhlcKO) mice display abnormally high bone mass and density and the BM cavity is severely occluded with bone (Figure 1A), accompanied by stunted B cell development, splenomegaly (Supplementary Figures 1A–E), and reduced BM cellularity compared to controls (Figure 1B)
Our results indicate that the oxygen levels in the microenvironment of Fraction A cells in the VhlcKO BM is similar to controls at 10weeks-old, but at 6-months-old, the microenvironment for Fraction A cells is relatively hypoxic compared to the microenvironments for Fraction B through Fraction F
Summary
The mechanisms by which changes in bone homeostasis affect immune development in the bone marrow (BM) are not fully understood [1–4]. A detailed understanding of how bone microenvironments affect immune cell development and function could provide strategies towards novel therapeutic approaches to immune deficiencies. VHL is expressed ubiquitously in many cell types, and global deletion of the Vhl gene results in embryonic lethality, so conditional knockout approaches are necessary to investigate the cellspecific roles of VHL in specific microenvironments. Conditional deletion of Vhl in OBs and in hematopoietic progenitors have demonstrated a role for VHL in these cell types [8, 9]. The role of HIF and its regulation on the immune system has been extensively reviewed [10], but the mechanisms by which cell-intrinsic and cell-extrinsic VHL regulate specific immune cell lineages has not fully been addressed
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