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

Probiotic <i>L. reuteri</i> Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Abstract Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 ( L. reuteri ) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri ‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014. © 2014 Wiley Periodicals, Inc.

Lactulose Suppresses Osteoclastogenesis and Ameliorates Estrogen Deficiency-Induced Bone Loss in Mice

<p>Postmenopausal osteoporosis is characterized by excess osteoclastogenesis which leads to net bone loss and brittle fractures. Studies have demonstrated that estrogen deficiency-associated bone loss is microbiota-dependent and could be prevented by probiotics and prebiotics. In this study, we report that orally administered lactulose (20 g/kg, 6 weeks) orally administered significantly inhibited osteoclastogenesis, bone resorption, and prevented ovariectomy (OVX)-induced bone loss in mice. Lactulose increased intestinal <i>Claudin 2, 3 and 15,</i> compared to the OVX group, and lowered pro-osteoclastogenic cytokines levels including tumor necrosis factor-α, interleukin(IL)-6, receptor activator of nuclear factor kappa-Β ligand (RANKL), and IL-17 as well as increased the anti-inflammatory cytokine IL-10 in the intestine, peripheral blood, and bone marrow. Lactulose significantly preserved the number of Foxp3⁺ Treg cells in the intestines compared with that in OVX mice. Lactulose altered the composition of intestinal microbiota measured by 16s rDNA sequencing and increased intestinal and serum short-chain fatty acids (SCFAs) levels including acetate, propionate and butyrate which were decreased in OVX mice as measured by gas chromatography. Oral administration of lactulose for 2 weeks significantly lowered the level of bone resorption marker C-telopeptide of type 1 collagen-1 in healthy male young volunteers (aging 20-25 years). In conclusion, lactulose inhibited osteoclastogenesis and bone resorption by altering the intestinal microbiota and increasing SCFAs. Lactulose could serve as an ideal therapeutic agent for postmenopausal osteoporosis.</p>

Probiotics Protect Mice from Ovariectomy-Induced Cortical Bone Loss

The gut microbiota (GM) modulates the hosts metabolism and immune system. Probiotic bacteria are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host and can alter the composition of the GM. Germ-free mice have increased bone mass associated with reduced bone resorption indicating that the GM also regulates bone mass. Ovariectomy (ovx) results in bone loss associated with altered immune status. The purpose of this study was to determine if probiotic treatment protects mice from ovx-induced bone loss. Mice were treated with either a single Lactobacillus (L) strain, L. paracasei DSM13434 (L. para) or a mixture of three strains, L. paracasei DSM13434, L. plantarum DSM 15312 and DSM 15313 (L. mix) given in the drinking water during 6 weeks, starting two weeks before ovx. Both the L. para and the L. mix treatment protected mice from ovx-induced cortical bone loss and bone resorption. Cortical bone mineral content was higher in both L. para and L. mix treated ovx mice compared to vehicle (veh) treated ovx mice. Serum levels of the resorption marker C-terminal telopeptides and the urinary fractional excretion of calcium were increased by ovx in the veh treated but not in the L. para or the L. mix treated mice. Probiotic treatment reduced the expression of the two inflammatory cytokines, TNFα and IL-1β, and increased the expression of OPG, a potent inhibitor of osteoclastogenesis, in cortical bone of ovx mice. In addition, ovx decreased the frequency of regulatory T cells in bone marrow of veh treated but not probiotic treated mice. In conclusion, treatment with L. para or the L. mix prevents ovx-induced cortical bone loss. Our findings indicate that these probiotic treatments alter the immune status in bone resulting in attenuated bone resorption in ovx mice.

Loss of Bone and Wnt10b Expression in Male Type 1 Diabetic Mice Is Blocked by the Probiotic Lactobacillus reuteri

Type 1 diabetes (T1D)–induced osteoporosis is characterized by a predominant suppression of osteoblast number and activity, as well as increased bone marrow adiposity but no change in osteoclast activity. The fundamental mechanisms and alternative anabolic treatments (with few side effects) for T1D bone loss remain undetermined. Recent studies by our laboratory and others indicate that probiotics can benefit bone health. Here, we demonstrate that Lactobacillus reuteri, a probiotic with anti-inflammatory and bone health properties, prevents T1D-induced bone loss and marrow adiposity in mice. We further found that L. reuteri treatment prevented the suppression of Wnt10b in T1D bone. Consistent with a role for attenuated bone Wnt10b expression in T1D osteoporosis, we observed that bone-specific Wnt10b transgenic mice are protected from T1D bone loss. To examine the mechanisms of this protection, we focused on TNF-α, a cytokine up-regulated in T1D that causes suppression of osteoblast Wnt10b expression in vitro. Addition of L. reuteri prevented TNF-α–mediated suppression of Wnt10b and osteoblast maturation markers. Taken together, our findings reveal a mechanism by which T1D causes bone loss and open new avenues for use of probiotics to benefit the bone.

Gut microbiota and bone metabolism

Abstract Osteoporosis is the most common metabolic skeletal disease. It is characterized by the deterioration of the skeletal microarchitecture and bone loss, leading to ostealgia, and even bone fractures. Accumulating evidence has indicated that there is an inextricable relationship between the gut microbiota (GM) and bone homeostasis involving host–microbiota crosstalk. Any perturbation of the GM can play an initiating and reinforcing role in disrupting the bone remodeling balance during the development of osteoporosis. Although the GM is known to influence bone metabolism, the mechanisms associated with these effects remain unclear. Herein, we review the current knowledge of how the GM affects bone metabolism in health and disease, summarize the correlation between pathogen‐associated molecular patterns of GM structural components and bone metabolism, and discuss the potential mechanisms underlying how GM metabolites regulate bone turnover. Deciphering the complicated relationship between the GM and bone health will provide new insights into the prevention and treatment of osteoporosis.

Effect of Bacillus subtilis C-3102 on bone mineral density in healthy postmenopausal Japanese women: a randomized, placebo-controlled, double-blind clinical trial

Gut microbiota influence the host immune system and are associated with various diseases. In recent years, postmenopausal bone loss has been suggested to be related to gut microbiota. In the present study, we investigated the treatment effect of the probiotic Bacillus subtilis C-3102 (C-3102) on bone mineral density (BMD) and its influence on gut microbiota in healthy postmenopausal Japanese women. Seventy-six healthy postmenopausal Japanese women were treated with a placebo or C-3102 spore-containing tablets for 24 weeks. When compared with the placebo, C-3102 significantly increased total hip BMD (placebo = 0.83 ± 0.63%, C-3102 = 2.53 ± 0.52%, p=0.043). There was a significant group-by-time interaction effect for urinary type I collagen cross-linked N-telopeptide (uNTx) (p=0.033), a marker of bone resorption. Specifically, the C-3102 group showed significantly lower uNTx when compared with the placebo group at 12 weeks of treatment (p=0.015). In addition, in the C-3102 group, there was a trend towards a decrease in the bone resorption marker tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) when compared with the placebo group at 12 weeks of treatment (p=0.052). The relative abundance of genus Bifidobacterium significantly increased at 12 weeks of treatment compared with the baseline in the C-3102 group. The relative abundance of genus Fusobacterium was significantly decreased in the C-3102 group at 12 and 24 weeks of treatment compared with the baseline. These data suggested that C-3102 improves BMD by inhibiting bone resorption and modulating gut microbiota in healthy postmenopausal women.

Montmorency tart cherry protects against age-related bone loss in female C57BL/6 mice and demonstrates some anabolic effects

Age-related bone loss is a consequence of endocrine and immune changes that disrupt bone remodeling. Functional foods (e.g., tart cherries) with antioxidant, anti-inflammatory and prebiotic activity can potentially counter this age-related phenomenon. The aim of this study was to determine if Montmorency tart cherry protects against early age-related bone loss and the culpable alterations in bone metabolism.

Probiotic <i>Lactobacillus reuteri</i> Prevents Postantibiotic Bone Loss by Reducing Intestinal Dysbiosis and Preventing Barrier Disruption

ABSTRACT   Antibiotic treatment, commonly prescribed for bacterial infections, depletes and subsequently causes long-term alterations in intestinal microbiota composition. Knowing the importance of the microbiome in the regulation of bone density, we investigated the effect of postantibiotic treatment on gut and bone health. Intestinal microbiome repopulation at 4-weeks postantibiotic treatment resulted in an increase in the Firmicutes:Bacteroidetes ratio, increased intestinal permeability, and notably reduced femoral trabecular bone volume (approximately 30%, p &lt; 0.01). Treatment with a mucus supplement (a high-molecular-weight polymer, MDY-1001 [MDY]) prevented the postantibiotic-induced barrier break as well as bone loss, indicating a mechanistic link between increased intestinal permeability and bone loss. A link between the microbiome composition and bone density was demonstrated by supplementing the mice with probiotic bacteria. Specifically, Lactobacillus reuteri, but not Lactobacillus rhamnosus GG or nonpathogenic Escherichia coli, reduced the postantibiotic elevation of the Firmicutes:Bacteroidetes ratio and prevented femoral and vertebral trabecular bone loss. Consistent with causing bone loss, postantibiotic-induced dysbiosis decreased osteoblast and increased osteoclast activities, changes that were prevented by both L. reuteri and MDY. These data underscore the importance of microbial dysbiosis in the regulation of intestinal permeability and bone health, as well as identify L. reuteri and MDY as novel therapies for preventing these adverse effects. © 2018 American Society for Bone and Mineral Research.

Estrogens and Androgens in Skeletal Physiology and Pathophysiology

Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution.

Estrogen and the skeleton

Estrogen is the major hormonal regulator of bone metabolism in women and men. Therefore, there is considerable interest in unraveling the pathways by which estrogen exerts its protective effects on bone. Although the major consequence of the loss of estrogen is an increase in bone resorption, estrogen deficiency is associated with a gap between bone resorption and formation, indicating that estrogen is also important for maintaining bone formation at the cellular level. Direct estrogen effects on osteocytes, osteoclasts, and osteoblasts lead to inhibition of bone remodeling, decreased bone resorption, and maintenance of bone formation, respectively.

Dried Plum’s Polyphenolic Compounds and Carbohydrates Contribute to Its Osteoprotective Effects and Exhibit Prebiotic Activity in Estrogen Deficient C57BL/6 Mice

Evidence of dried plum’s benefits on bone continues to emerge. This study investigated the contribution of the fruit’s polyphenol (PP) and carbohydrate (CHO) components on a bone model of postmenopausal osteoporosis to explore their prebiotic activity. Osteopenic ovariectomized mice were fed diets supplemented with dried plum, a crude extract of dried plum’s polyphenolic compounds, or the PP or CHO fraction of the crude extract. The effects of treatments on the bone phenotype were assessed at 5 and 10 weeks as well as the prebiotic activity of the different components of dried plum. Both the CHO and PP fractions of the extract contributed to the effects on bone with the CHO suppressing bone formation and resorption, and the PP temporally down-regulating formation. The PP and CHO components also altered the gut microbiota and cecal short chain fatty acids. These findings demonstrate that the CHO as well as the PP components of dried plum have potential prebiotic activity, but they have differential roles in mediating the alterations in bone formation and resorption that protect bone in estrogen deficiency.

Bone Remodeling in Post-menopausal Osteoporosis

Bone mass in the skeleton is dependent on the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts in discrete bone multi-cellular units. Remodeling of bone in these units is important not only for maintaining bone mass, but also to repair microdamage, to prevent accumulation of too much old bone, and for mineral homeostasis. The activities of osteoblasts and osteoclasts are controlled by a variety of hormones and cytokines, as well as by mechanical loading. Most importantly, sex hormones are very crucial for keeping bone mass in balance, and the lack of either estrogen or testosterone leads to decreased bone mass and increased risk for osteoporosis. The prevalence of osteoporotic fractures is increasing dramatically in the Western part of the world and is a major health problem in many countries. In the present review, the cellular and molecular mechanisms controlling bone remodeling and the influence of sex hormones on these processes are summarized. In a separate paper in this issue, the pathogenesis of post-menopausal osteoporosis will be compared with that of inflammation-induced bone remodeling, including the evidence for and against the hypothesis that concomitant post-menopausal osteoporotic disease influences the progression of periodontal disease.

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

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.

The gut microbiota in osteoarthritis: where do we stand and what can we do?

Osteoarthritis (OA) is one of the most frequent musculoskeletal diseases characterized by degeneration of articular cartilage, subchondral bone remodeling, and synovial membrane inflammation, which is a leading cause of global disability, morbidity, and decreased quality of life. Interpreting the potential mechanisms of OA pathogenesis is essential for developing novel prevention and disease-modifying therapeutic interventions. Gut microbiota is responsible for a series of metabolic, immunological, and structural and neurological functions, potentially elucidating the heterogeneity of OA phenotypes and individual features. In this narrative review, we summarized research evidence supporting the hypothesis of a “gut-joint axis” and the interaction between gut microbiota and the OA-relevant factors, including age, gender, genetics, metabolism, central nervous system, and joint injury, elucidating the underlying mechanisms of this intricate interaction. In the context, we also speculated the promising manipulation of gut microbiota in OA management, such as exercise and fecal microbiota transplantation (FMT), highlighting the clinical values of gut microbiota. Additionally, future research directions, such as more convincing studies by the interventions of gut microbiota, the gene regulation of host contributing to or attributed to the specific phenotypes of gut microbiota related to OA, and the relevance of distinct cell subgroups to gut microbiota, are expected. Moreover, gut microbiota is also the potential biomarker related to inflammation and gut dysbiosis that is able to predict OA progression and monitor the efficacy of therapeutic intervention.

The gut microbiota regulates bone mass in mice

Abstract The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4+ T cells and CD11b+/GR 1 osteoclast precursor cells in bone marrow, which could be normalized by colonization. GF mice exhibited reduced expression of inflammatory cytokines in bone and bone marrow compared with CONV-R mice. In summary, the gut microbiota regulates bone mass in mice, and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-mediated bone resorption. Further studies are required to evaluate the gut microbiota as a novel therapeutic target for osteoporosis. © 2012 American Society for Bone and Mineral Research.

Associations Between Inflammatory Mediators and Bone Outcomes in Postmenopausal Women: A Cross-Sectional Analysis of Baseline Data from the Prune Study

These findings suggest that inflammatory markers may be an important mediator for postmenopausal bone loss.