Genetic factors account for the majority of the variance of human bone mass, but the contribution of non-genetic factors remains largely unknown. By utilizing maternal/offspring transmission, cohabitation, or fecal material transplantation (FMT) studies, we investigated the influence of the gut microbiome on skeletal maturation. We show that the gut microbiome is a communicable regulator of bone structure and turnover in mice. In addition, we found that the acquisition of a specific bacterial strain, segmented filamentous bacteria (SFB), a gut microbe that induces intestinal Th17 cell expansion, was sufficient to negatively impact skeletal maturation. These findings have significant translational implications, as the identification of methods or timing of microbiome transfer may lead to the development of bacteriotherapeutic interventions to optimize skeletal maturation in humans. Moreover, the transfer of SFB-like microbes capable of triggering the expansion of human Th17 cells during therapeutic FMT procedures could lead to significant bone loss in fecal material recipients.

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.

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.

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.

Primary hyperparathyroidism (PHPT) is a common cause of bone loss that is modeled by continuous PTH (cPTH) infusion. Here we show that the inflammatory cytokine IL-17A is upregulated by PHPT in humans and cPTH in mice. In humans, IL-17A is normalized by parathyroidectomy. In mice, treatment with anti-IL-17A antibody and silencing of IL-17A receptor IL-17RA prevent cPTH-induced osteocytic and osteoblastic RANKL production and bone loss. Mechanistically, cPTH stimulates conventional T cell production of TNFα (TNF), which increases the differentiation of IL-17A-producing Th17 cells via TNF receptor 1 (TNFR1) signaling in CD4+ cells. Moreover, cPTH enhances the sensitivity of naive CD4+ cells to TNF via GαS/cAMP/Ca2+ signaling. Accordingly, conditional deletion of GαS in CD4+ cells and treatment with the calcium channel blocker diltiazem prevents Th17 cell expansion and blocks cPTH-induced bone loss. Neutralization of IL-17A and calcium channel blockers may thus represent novel therapeutic strategies for hyperparathyroidism.

Hydrogen sulfide (H2S) is a gasotransmitter known to regulate bone formation and bone mass in unperturbed mice. However, it is presently unknown whether H2S plays a role in pathologic bone loss. Here we show that ovariectomy (ovx), a model of postmenopausal bone loss, decreases serum H2S levels and the bone marrow (BM) levels of two key H2S-generating enzymes, cystathione β-synthase (CBS) and cystathione γ-lyase (CSE). Treatment with the H2S-donor GYY4137 (GYY) normalizes serum H2S in ovx mice, increases bone formation, and completely prevents the loss of trabecular bone induced by ovx. Mechanistic studies revealed that GYY increases murine osteoblastogenesis by activating Wnt signaling through increased production of the Wnt ligands Wnt16, Wnt2b, Wnt6, and Wnt10b in the BM. Moreover, in vitro treatment with 17β-estradiol upregulates the expression of CBS and CSE in human BM stromal cells (hSCs), whereas an H2S-releasing drug induces osteogenic differentiation of hSCs. In summary, regulation of H2S levels is a novel mechanism by which estrogen stimulates osteoblastogenesis and bone formation in mice and human cells. Blunted production of H2S contributes to ovx-induced bone loss in mice by limiting the compensatory increase in bone formation elicited by ovx. Restoration of H2S levels is a potential novel therapeutic approach for postmenopausal osteoporosis.

Bone loss is a frequent but not universal complication of hyperparathyroidism. Using antibiotic-treated or germ-free mice, we show that parathyroid hormone (PTH) only caused bone loss in mice whose microbiota was enriched by the Th17 cell-inducing taxa segmented filamentous bacteria (SFB). SFB+ microbiota enabled PTH to expand intestinal TNF+ T and Th17 cells and increase their S1P-receptor-1 mediated egress from the intestine and recruitment to the bone marrow (BM) that causes bone loss. CXCR3-mediated TNF+ T cell homing to the BM upregulated the Th17 chemoattractant CCL20, which recruited Th17 cells to the BM. This study reveals mechanisms for microbiota-mediated gut–bone crosstalk in mice models of hyperparathyroidism that may help predict its clinical course. Targeting the gut microbiota or T cell migration may represent therapeutic strategies for hyperparathyroidism.

Tyagi et al. show that oral supplementation with the widely used probiotic, Lactobacillus rhamnosus GG (LGG), increases bone mass in mice by increasing the serum levels of the short chain fatty acid butyrate. LGG or butyrate increase the frequency of regulatory T (Treg) cells in the intestine and in the bone marrow. Treg cells stimulate CD8 + T cells to secrete the Wnt ligand Wnt10b, which stimulates bone formation by activating Wnt signaling in osteoblasts. Therefore, LGG and butyrate may represent new interventions for the prevention and treatment of osteoporosis.

T cells are required for continuous PTH (cPTH) treatment to induce bone loss as they sensitize SCs to PTH through CD40 Ligand (CD40L), a surface molecule of activated T cells. Since CD40L expression is a feature of activated T cells, we investigated whether antigen (Ag) mediated T cell activation is required for PTH to exert its catabolic activity. We report that inhibition of Ag presentation through silencing of either class I or class II MHC-T cell receptor (TCR) interaction prevents the cortical bone loss induced by in vivo cPTH treatment. We also show that the bone loss and the stimulation of bone resorption induced by cPTH treatment are prevented by CTLA4-Ig, an inhibitor of T cell costimulation approved for the treatment of Rheumatoid Arthritis. Since inhibition of antigen driven T cell activation by blockade of either TCR signaling or T cell costimulation is sufficient to silence the catabolic activity of cPTH, antigen presenting cells and T lymphocyte interactions therefore play a critical role in the mechanism of action of PTH.

PTH promotes bone catabolism by targeting bone marrow stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 Ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, RANKL/OPG production and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, lifespan and function through CD40L. T cell-SC cross-talk pathways may thus provide pharmacological targets for PTH induced bone disease.