Mechanism of action of gut microbiota and probiotic Lactobacillus rhamnosus GG on skeletal remodeling in mice.

Introduction: Gut microbiota (GM) is the collection of small organisms such as bacteria, fungi, bacteriophages and protozoans living in the intestine in symbiotics relation within their host. GM regulates host metabolism by various mechanisms.

Methods: This review aims to consolidate current information for physicians on the effect of GM on bone health.

Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown.

Parathyroid hormone-dependent bone formation requires butyrate production by intestinal microbiota.

Parathyroid hormone (PTH) is a critical regulator of skeletal development that promotes both bone formation and bone resorption. Using microbiota depletion by wide-spectrum antibiotics and germ-free (GF) female mice, we showed that the microbiota was required for PTH to stimulate bone formation and increase bone mass. Microbiota depletion lowered butyrate levels, a metabolite responsible for gut-bone communication, while reestablishment of physiologic levels of butyrate restored PTH-induced anabolism.

The Microbial Metabolite Butyrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT10B Expression.

Nutritional supplementation with probiotics can prevent pathologic bone loss. Here we examined the impact of supplementation with Lactobacillus rhamnosus GG (LGG) on bone homeostasis in eugonadic young mice. Micro-computed tomography revealed that LGG increased trabecular bone volume in mice, which was due to increased bone formation.

IL-17 Receptor Signaling in Osteoblasts/Osteocytes Mediates PTH-Induced Bone Loss and Enhances Osteocytic RANKL Production.

Primary hyperparathyroidism (PHPT) is a condition where elevated PTH levels lead to bone loss, in part through increased production of the osteoclastogenic factor IL-17A, by bone marrow (BM) T-helper 17 (Th17) cells, a subset of helper CD4+ T cells. In animals, PHPT is modeled by continuous PTH treatment (cPTH). In mice, an additional critical action of cPTH is the capacity to increase the production of RANKL by osteocytes.

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 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.

3-Piperidylethoxypterocarpan: A potential bone anabolic agent that improves bone quality and restores trabecular micro-architecture in ovariectomized osteopenic rats.

A series of new 6H-benzofuro[3, 2-c]chromenes (BFC, pterocarpans) with structure-activity relationships were investigated for their potential use in osteoporosis treatment. One of the BFCs 3-piperidylethoxypterocarpan 20 promotes osteoblast differentiation and mineralization at a dose as low as 1 pM via activation of ER/P38MAPK/BMP-2 pathway. When evaluated for in-vivo osteogenic activity in female Sprague-Dawley rats, BFC 20 increased bone mineral density and new bone formation, compared with control at 1.

IL-18BP is decreased in osteoporotic women: Prevents Inflammasome mediated IL-18 activation and reduces Th17 differentiation.

IL-18BP is a natural antagonist of pro-inflammatory IL-18 cytokine linked to autoimmune disorders like rheumatoid arthritis. However, its role in post menopausal osteoporosis is still unknown. In this study, we investigated the role of IL-18BP on murine osteoblasts, its effect on osteoblasts-CD4+ T cells and osteoblasts-CD11b+ macrophage co-culture.

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.

Methoxyisoflavones formononetin and isoformononetin inhibit the differentiation of Th17 cells and B-cell lymphopoesis to promote osteogenesis in estrogen-deficient bone loss conditions.

Objective: Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model.

Hydrogen Sulfide Is a Novel Regulator of Bone Formation Implicated in the Bone Loss Induced by Estrogen Deficiency.

Hydrogen sulfide (H2 S) is a gasotransmitter known to regulate bone formation and bone mass in unperturbed mice. However, it is presently unknown whether H2 S plays a role in pathologic bone loss. Here we show that ovariectomy (ovx), a model of postmenopausal bone loss, decreases serum H2 S levels and the bone marrow (BM) levels of two key H2 S-generating enzymes, cystathione β-synthase (CBS) and cystathione γ-lyase (CSE).

IL-17A Is Increased in Humans with Primary Hyperparathyroidism and Mediates PTH-Induced Bone Loss in Mice.

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.

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.

Enhanced immunoprotective effects by anti-IL-17 antibody translates to improved skeletal parameters under estrogen deficiency compared with anti-RANKL and anti-TNF-α antibodies.

Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF-κB ligand (RANKL), tumor necrosis factor α (TNF-α), and interleukin 17 (IL-17), all of which augment osteoclastogenesis. RANKL and TNF-α are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF-α.

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.

Greater Skeletal Gains in Ovary Intact Rats at Maturity Are Achieved by Supplementing a Standardized Extract of Butea monosperma Stem Bark that Confers Better Bone Conserving Effect following Ovariectomy and Concurrent Treatment Withdrawal.

With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of Butea monosperma resulted in maximizing bone gain in rats during growth and maturation and thus protected against osteopenia following ovariectomy (OVx) with concomitant treatment withdrawal. Female rats at weaning were given ASF (100 mg/kg/d) or vehicle for 12 weeks, and baseline skeletal parameters (micro-CT) and total plasma antioxidant status (TAS) were measured. At this stage, one group was OVx and the other group was sham operated.

Estrogen deficiency induces the differentiation of IL-17 secreting Th17 cells: a new candidate in the pathogenesis of osteoporosis.

Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss.

Formononetin reverses established osteopenia in adult ovariectomized rats.

Objective: Formononetin (Formo) prevents ovariectomy (Ovx)-induced bone loss in rats. However, there are no reports on the curative effects of Formo. The objective of this study was to investigate the ability of Formo in restoring trabecular microarchitecture and promoting new bone formation in osteopenic rats.

Daidzein prevents the increase in CD4+CD28null T cells and B lymphopoesis in ovariectomized mice: a key mechanism for anti-osteoclastogenic effect.

Estrogen deficiency leads to an upregulation of TNF-α producing T cells and B-lymphopoesis which augments osteoclastogenesis. Estrogen deficiency also increases the population of premature senescent CD4⁺ CD28null T cells which secrete a higher amount of TNF-α thus leading to enhanced osteoclastogenesis. Isoflavonoids like daidzein and genistein are found mostly in soybeans, legumes, and peas.

Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway.