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    • Despite the dramatic changes in

    2018-10-31

    Despite the dramatic changes in bone morphology in OsxCre;iDTR mutants, bone marrow cellularity was unaffected compared with their control littermates (Figure 3A). Red blood cell and platelet numbers were unchanged in the blood of the OsxCre;iDTR mutant, but there was a 50% decrease in the white blood cell count due to severe lymphopenia (Figure 3B). The mutant bone marrow had fewer mature B220+IgM+ B cells, increased monocytes and granulocytes (Figure 3A), and a corresponding increase in granulocyte and macrophage progenitors (GMPs) (Figure 3C). Given the importance of the spleen for B lymphopoiesis and extramedullary hematopoiesis, we analyzed the spleens and noted no difference in weight, but similarly increased Mac1+Gr1+ cells in the OsxCre;iDTR mutants (Figure 3D). In summary, Osx cell deletion caused a loss of mature ACA and an increase in myeloid cells in both the bone marrow and spleen of mutant animals. We assessed whether the GMP increase in the OsxCre;iDTR mutants was secondary to increased proliferation or decreased death of GMPs. There was change in neither cell-cycle status (Figure S3A) nor apoptosis (Figure S3B). The accumulation of GMP and Mac1+Gr1+ cells could either be an osteoprogenitor-mediated hematopoietic effect or a consequence of inflammatory phagocytic-type immune response triggered by the death of osteolineage cells. To test this hypothesis, we co-injected indomethacin, a non-steroidal anti-inflammatory drug, together with DT in OsxCre;DTR mutant mice for 1 week. Bone marrow analysis revealed normalization of the GMP, Mac1+, and Mac1+Gr1+ cell populations in indomethacin-treated animals (Figure S3C). Therefore, the increase in Mac1+Gr1+ myeloid cells could be due to inflammatory response to osteolineage cell death, although we cannot exclude an osteolineage cell-mediated myeloid effect. We asked whether HSC function was affected by the short-term deletion of Osx cells. We observed no differences between the OsxCre;iDTR mutants and controls in long-term HSC (LINloC-KIT+SCA-1+CD48−CD150+) number (Figure S4A), proliferation (Figure S4B), or apoptosis (Figure S4C). There was an increase in LINloC-KIT+SCA-1− progenitor cell number and cell cycle (Figures S4A and S4B). However, no defect in HSC function was detected when bone marrow cells from were transplanted into primary and secondary recipients in a 1:1 ratio with congenic SJL competitor cells (Figure S4D). These data suggest that short-term deletion of Osx cells did not affect HSC function. Given the diminished production of mature B cells, we assessed CLPs and found no difference (Figure 3C). However, intermediate stages of B cell development were affected. Specifically, a decrease in both pre-B and mature B populations were noted while pro-B cells, especially during the later C′ pro-B and C″ pro-B stages, were significantly increased (Figure 4A). These data strongly suggest that B cell differentiation was impaired at the pro-B to pre-B juncture. We next tested whether these changes affected the animal\'s immune response. Mice were challenged with NP-Ficoll to trigger a T cell-independent (Maizels et al., 1988) immune response and followed for 28 days. Data revealed that OsxCre;iDTR mutants could not sustain immunoglobulin G (IgG) and M (IgM) production over time compared with their control littermates (Figures 4B and 4C). These data indicate that the perturbation in B cell development was not simply immunophenotypic, but there was also a functional reduction in Ig production and immune response. To determine whether the B cell differentiation defect was microenvironment-dependent, we transplanted 1 × 106 bone marrow cells from congenic SJL mice into lethally irradiated control or mutant OsxCre;iDTR recipients. A similar rate of reconstitution was seen in both control and mutants (Figure 5A). As wild-type SJL cells repopulated both control and mutant OsxCre;iDTR recipients over the next 12 weeks, we observed a gradual recapitulation of the OsxCre+/−;DTRFl/+ hematopoietic phenotype in the mutant recipients but not in the controls (Figures 5B–5F). These data strongly suggest that the incomplete B cell differentiation observed in OsxCre;iDTR was caused by an altered bone microenvironment.