Deletion Of The Von Hippel-Lindau Gene Interferes With The Development Of Peripheral B-Cell Subsets

Abstract

The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes.In order to study the role of pVhl in hematopoiesis we crossed Vhl conditional mice with vav-iCre mice to induce a constitutive hematopoiesis-specific Vhl deletion (VhlKOvav). As expected, we observed a dramatic expansion of spleen erythropoiesis, however bone marrow lin-Sca-1+c-kit+CD48-CD150+ hematopoietic stem cell numbers of VhlKOvav mice did not significantly differ from control mice. The most striking observation in VhlKOvav mice was that B-cell numbers in bone marrow and spleen were decreased by 53% and 78%, respectively. In order to exclude that the loss of B-cells in VhlKOvav mice was due to a B-cell extrinsic effect we crossed Vhl conditional mice to the B-cell specific deleter lines CD19-Cre and Mb1-Cre (VhlKOCD19 and VhlKOMb1 mice). Flow cytometric analysis also revealed decreased peripheral B-cell numbers in VhlKOCD19 mice and an even more pronounced B-cell loss in VhlKOMb1 mice (B-cells spleen, mean±SEM; control (n=9), 77±2.4x106; VhlKOMb1 (n=12), 1.3±0.2x106; p<0.001). This demonstrated that pVhl is cell-autonomously required for the normal development of the B-cell compartment. To more accurately define the Vhl-null B-cell developmental defect we analyzed bone marrow B-cell subsets of VhlKOMb1 and control mice. Early B-cell progenitor numbers defined by the surface markers B220, CD43, CD24, BP-1 (Hardy's classification, fractions A-D) of VhlKOMb1 mice were not altered. In contrast, we observed a significant 41% reduction of the immature B220+CD43-IgM+IgD- and a 46% reduction of the mature B220+CD43-IgM+IgD+ bone marrow VhlKOMb1 B-cell numbers compared to controls. In peripheral blood VhlKOMb1 immature and mature B-cell numbers were even more decreased (by 81% and 86%, respectively). We hypothesized that increased CXCR4 expression, which is negatively regulated by pVhl, could be responsible for the decreased bone marrow egress of immature bone marrow B-cells. Indeed, we observed a more than twofold increase of CXCR4 expression of VhlKOMb1 compared to control bone marrow immature B-cells (MFI, mean±SEM; control (n=3), 72±15; VhlKOMb1 (n=3), 162±23; p<0.05). Strikingly, VhlKOMb1 spleens were almost devoid of follicular B220+CD21/35intmCD23+IgD+IgM+ and marginal zone B220+CD21/35highCD23-IgMhigh B-cells and accordingly follicular structures could not be observed in histological sections. In VhlKOMb1 lymph nodes the follicular B-cell numbers were also dramatically decreased. Next, we flow sorted residual splenic VhlKOMb1 B-cells and were able to confirm deletion of the Vhl-gene by PCR. Target genes of the hypoxic-response pathway such as Pgk1, Vegf and Bnip3 were 10- to more than 100-fold higher expressed in sorted VhlKOMb1 compared to control B-cells. As a possible reason for the low VhlKOMb1 peripheral B-cell numbers we identified a more than twofold reduction in CD62L expression by immature blood B-cells (MFI, mean±SEM; control (n=3), 3127±250; VhlKOMb1 (n=3), 1528±66; p<0.05) which presumably impaired their homing ability to peripheral lymphoid organs. Additionally, we detected an increased B-cell apoptosis rate of VhlKOMb1 B-cells in the spleen. Finally, we were able to show that decreased follicular splenic B-cell numbers of VhlKOCD19 mice could be completely rescued by additionally breeding Hif-1α conditional alleles into the system (Hif-1αVhlKOCD19mice).In summary, we identified pVhl as a key regulator of peripheral B-cell maturation. We show that pVhl-mediated negative regulation of the hypoxic-response pathway is required for normal peripheral B-cell differentiation. Our data suggest that B-cell pVHL loss-of-function leads to decreased bone marrow egress and decreased lymphoid organ homing of immature B-cells mediated by the dysregulation of CXCR4 and CD62L. Disclosures:No relevant conflicts of interest to declare.