Annette I. Garbe

A novel resorption assay for osteoclast functionality based on an osteoblast-derived native extracellular matrix

Osteoclasts are large, mobile, bone‐resorbing cells and play a critical role in bone remodeling and catabolic bone diseases. The major function of osteoclasts is to hydrolyze inorganic hydroxyapatite and degrade organic bone matrix, mainly collagen. For evaluation of differentiation to fully functional osteoclasts in vitro, a quantitative functional resorption assay is essential. Currently available commercial test systems are either based on the organic or the inorganic part of the bone matrix. The novel resorption assay presented here is based on decellularized osteoblast‐derived matrix. SaOS‐2 cells were used for the synthesis of a densely mineralized extracellular bone matrix (ECM) in α‐MEM medium, which strongly accelerates their matrix synthesis. After removal of the SaOS‐2 cells, osteoclast precursors are plated on the osteoblast‐derived matrix and stained by von Kossa. Subsequently, resorption pits were quantified by densitometry using an imaging program. Using this novel assay, we show that (i) RAW 264.7 cells resorbed the osteoblast‐derived matrix continuously from day 6 until day 9 of culture, a process that is dose dependent on the macrophage colony‐stimulating factor (M‐CSF) concentration, (ii) the resorption performance of RAW 264.7 was dose‐dependently inhibited by IFN‐γ, and (iii) the assay is working with primary human and mouse osteoclast precursors as well. In conclusion, this quantitative, functional, easy‐to‐use, inexpensive assay will advance analysis of osteoclast biology. J. Cell. Biochem. 109: 1025–1032, 2010.

αβ versus γδ fate choice: counting the T-cell lineages at the branch point

Summary:  Both αβ and γδ T cells develop in the thymus from a common progenitor. Historically distinguished by their T‐cell receptor (TCR), these lineages are now defined on the basis of distinct molecular programs. Intriguingly, in many transgenic and knockout systems these programs are mismatched with the TCR type, leading to the development of γδ lineage cells driven by αβTCR and vice versa. These puzzling observations were recently explained by the demonstration that TCR signal strength, rather than TCR type per se, instructs lineage fate, with stronger TCR signal favoring γδ and weaker signal favoring αβ lineage fates. These studies also highlighted the ERK (extracellular signal regulated kinase)‐Egr (early growth response)‐Id3 (inhibitor of differentiation 3) axis as a potential molecular switch downstream of TCR that determines lineage choice. Indeed, removal of Id3 was sufficient to redirect TCRγδ transgenic cells to the αβ lineage, even in the presence of strong TCR signal. However, in TCR non‐transgenic Id3 knockout mice the overall number of γδ lineage cells was increased due to an outgrowth of a Vγ1Vδ6.3 subset, suggesting that not all γδ T cells depend on this molecular switch for lineage commitment. Thus, the γδ lineage may in fact be a collection of two or more lineages not sharing a common molecular program and thus equipollent to the αβ lineage. TCR signaling is not the only factor that is required for development of αβ and γδ lineage cells; other pathways, such as signaling from Notch and CXCR4 receptors, cooperate with the TCR in this process.

Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70

Bone remodeling involves tightly regulated bone‐resorbing osteoclasts and bone‐forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F‐actin binding and regulatory protein SWAP‐70 in osteoclast biology. F‐actin ring formation, cell morphology, and bone resorption are impaired in Swap‐70−/− osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) remains unaffected. Swap‐70−/− mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP‐70 in Swap‐70−/− osteoclasts in vitro rescues their deficiencies in bone resorption and F‐actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP‐70, and the F‐actin binding domain. Transplantation of SWAP‐70–proficient bone marrow into Swap‐70−/− mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP‐70 in promoting osteoclast function through modulating membrane‐proximal F‐actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis.

Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy

The role of Foxp3-expressing regulatory T (Treg) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of Treg cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of Treg cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive Treg cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3+ Treg cells.

SWEF Proteins Distinctly Control Maintenance and Differentiation of Hematopoietic Stem Cells

SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner.

The F-actin modulator SWAP-70 controls podosome patterning in osteoclasts

Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclasts resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70−/− osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70−/− osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70s F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts.

Induced B cell development in adult mice

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.