Jau-Yi Li

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand

The bone loss induced by ovariectomy (ovx) has been linked to increased production of osteoclastogenic cytokines by bone marrow cells, including T cells and stromal cells (SCs). It is presently unknown whether regulatory interactions between these lineages contribute to the effects of ovx in bone, however. Here, we show that the T-cell costimulatory molecule CD40 ligand (CD40L) is required for ovx to expand SCs; promote osteoblast proliferation and differentiation; regulate the SC production of the osteoclastogenic factors macrophage colony-stimulating factor, receptor activator of nuclear factor-κB ligand, and osteoprotegerin; and up-regulate osteoclast formation. CD40L is also required for ovx to activate T cells and stimulate their production of TNF. Accordingly, ovx fails to promote bone loss and increase bone resorption in mice depleted of T cells or lacking CD40L. Therefore, cross-talk between T cells and SCs mediated by CD40L plays a pivotal role in the disregulation of osteoblastogenesis and osteoclastogenesis induced by ovx.

Sex steroid deficiency–associated bone loss is microbiota dependent and prevented by probiotics

A eubiotic microbiota influences many physiological processes in the metazoan host, including development and intestinal homeostasis. Here, we have shown that the intestinal microbiota modulates inflammatory responses caused by sex steroid deficiency, leading to trabecular bone loss. In murine models, sex steroid deficiency increased gut permeability, expanded Th17 cells, and upregulated the osteoclastogenic cytokines TNFα (TNF), RANKL, and IL-17 in the small intestine and the BM. In germ-free (GF) mice, sex steroid deficiency failed to increase osteoclastogenic cytokine production, stimulate bone resorption, and cause trabecular bone loss, demonstrating that the gut microbiota is central in sex steroid deficiency-induced trabecular bone loss. Furthermore, we demonstrated that twice-weekly treatment of sex steroid-deficient mice with the probiotics Lactobacillus rhamnosus GG (LGG) or the commercially available probiotic supplement VSL#3 reduces gut permeability, dampens intestinal and BM inflammation, and completely protects against bone loss. In contrast, supplementation with a nonprobiotic strain of E. coli or a mutant LGG was not protective. Together, these data highlight the role that the gut luminal microbiota and increased gut permeability play in triggering inflammatory pathways that are critical for inducing bone loss in sex steroid-deficient mice. Our data further suggest that probiotics that decrease gut permeability have potential as a therapeutic strategy for postmenopausal osteoporosis.

Silencing of parathyroid hormone (PTH) receptor 1 in T cells blunts the bone anabolic activity of PTH

Intermittent parathyroid hormone (iPTH) treatment stimulates T-cell production of the osteogenic Wnt ligand Wnt10b, a factor required for iPTH to activate Wnt signaling in osteoblasts and stimulate bone formation. However, it is unknown whether iPTH induces Wnt10b production and bone anabolism through direct activation of the parathyroid hormone (PTH)/PTH-related protein receptor (PPR) in T cells. Here, we show that conditional silencing of PPR in T cells blunts the capacity of iPTH to induce T-cell production of Wnt10b; activate Wnt signaling in osteoblasts; expand the osteoblastic pool; and increase bone turnover, bone mineral density, and trabecular bone volume. These findings demonstrate that direct PPR signaling in T cells plays an important role in PTH-induced bone anabolism by promoting T-cell production of Wnt10b and suggest that T cells may provide pharmacological targets for bone anabolism.

Disruption of PTH Receptor 1 in T Cells Protects against PTH-Induced Bone Loss

Background Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs. Methodology/Principal Findings Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor α (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio. Conclusions/Significance These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism.

PTH expands short-term murine hemopoietic stem cells through T cells

Intermittent parathyroid hormone (iPTH) treatment expands hemopoietic stem and progenitor cells (HSPCs), but the involved mechanisms and the affected HSPC populations are mostly unknown. Here we show that T cells are required for iPTH to expand short-term HSPCs (ST-HSPCs) and improve blood cell engraftment and host survival after BM transplantation. Silencing of PTH/PTH-related protein receptor (PPR) in T cells abrogates the effects of iPTH, thus demonstrating a requirement for direct PPR signaling in T cells. Mechanistically, iPTH expands ST-HSPCs by activating Wnt signaling in HSPCs and stromal cells (SCs) through T-cell production of the Wnt ligand Wnt10b. Attesting to the relevance of Wnt10b, iPTH fails to expand ST-HSPCs in mice with Wnt10b(-/-) T cells. Moreover, iPTH fails to promote engraftment and survival after BM transplantation in Wnt10b null mice. In summary, direct PPR signaling in T cells and the resulting production of Wnt10b play a pivotal role in the mechanism by which iPTH expands ST-HSPCs. The data suggest that T cells may provide pharmacologic targets for HSPC expansion.

The Sclerostin-Independent Bone Anabolic Activity of Intermittent PTH Treatment Is Mediated by T-Cell–Produced Wnt10b

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T‐cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti‐Scl antibody (Scl‐Ab) to determine the contribution of T‐cell–produced Wnt10b to the Scl‐independent modalities of action of iPTH. We report that combined treatment with Scl‐Ab and iPTH was more potent than either iPTH or Scl‐Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T‐cell–null mice and mice lacking T‐cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl‐Ab alone. However, in these mice, combined treatment with Scl‐Ab and iPTH was equally effective as Scl‐Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl‐independent activity of iPTH on osteoblasts and bone mass is mediated by T‐cell–produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl‐Ab than either alone.

Ovariectomy expands murine short-term hemopoietic stem cell function through T cell expressed CD40L and WNT10B

Estrogen deficiency expands hemopoietic stem and progenitor cells (HSPCs) and mature blood lineages, but the involved mechanism and the affected HSPC populations are mostly unknown. Here we show that ovariectomy (ovx) expands short-term HSPCs (ST-HSPCs) and improves blood cell engraftment and host survival after bone marrow (BM) transplantation through a dual role of the T-cell costimulatory molecule CD40 ligand (CD40L). This surface receptor is required for ovx to stimulate T-cell production of Wnt10b, a Wnt ligand that activates Wnt signaling in HSPCs and stromal cells (SCs). Moreover, CD40L is required for ovx to increase SC production of the hemopoietic cytokines interleukin (IL)-6, IL-7, and granulocyte macrophage-colony-stimulating factor. Attesting to the relevance of CD40L and Wnt10b, ovx fails to expand ST-HSPCs in CD40L-null mice and in animals lacking global or T-cell expression of Wnt10b. In summary, T cells expressed CD40L, and the resulting increased production of Wnt10b and hemopoietic cytokines by T cells and SCs, respectively, plays a pivotal role in the mechanism by which ovx regulates hemopoiesis. The data suggest that antiestrogens may represent pharmacological targets to improve ST-HSPC function through activation of the microenvironment.

T Cell–Expressed CD40L Potentiates the Bone Anabolic Activity of Intermittent PTH Treatment

T cells are known to potentiate the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. One of the involved mechanisms is increased T cell secretion of Wnt10b, a potent osteogenic Wnt ligand that activates Wnt signaling in stromal cells (SCs). However, additional mechanisms might play a role, including direct interactions between surface receptors expressed by T cells and SCs. Here we show that iPTH failed to promote SC proliferation and differentiation into osteoblasts (OBs) and activate Wnt signaling in SCs of mice with a global or T cell–specific deletion of the T cell costimulatory molecule CD40 ligand (CD40L). Attesting to the relevance of T cell–expressed CD40L, iPTH induced a blunted increase in bone formation and failed to increase trabecular bone volume in CD40L–/– mice and mice with a T cell–specific deletion of CD40L. CD40L null mice exhibited a blunted increase in T cell production of Wnt10b and abrogated CD40 signaling in SCs in response to iPTH treatment. Therefore, expression of the T cell surface receptor CD40L enables iPTH to exert its bone anabolic activity by activating CD40 signaling in SCs and maximally stimulating T cell production of Wnt10b.

Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH‐induced bone anabolism in mice

Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration, but the cellular and molecular mechanisms of action of iPTH are largely unknown. Here, we show that Teriparatide and iPTH cause a ~two‐threefold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents the increase in bone formation and trabecular bone volume and structure induced by iPTH. Therefore, increasing the number of Tregs is a pivotal mechanism by which iPTH exerts its bone anabolic activity. Increasing Tregs pharmacologically may represent a novel bone anabolic therapy, while iPTH‐induced Treg increase may find applications in inflammatory conditions and transplant medicine.