Lactobacillus Reuteri 6475 Prevents Bone Loss in a Clinically Relevant Oral Model of Glucocorticoid-Induced Osteoporosis in Male CD-1 Mice.

Glucocorticoids (GCs) are commonly used anti-inflammatory medications with significant side effects, including glucocorticoid-induced osteoporosis (GIO). We have previously demonstrated that chronic subcutaneous GC treatment in mice leads to gut barrier dysfunction and trabecular bone loss. We further showed that treating with probiotics or barrier enhancers improves gut barrier function and prevents GIO. The overall goal of this study was to test if probiotics could prevent GC-induced gut barrier dysfunction and bone loss in a clinically relevant oral-GC model of GIO. Eight-week-old male CD-1 mice were treated with vehicle or corticosterone in the drinking water for 4 weeks and administered probiotics Lactobacillus reuteri ATCC 6475 (LR 6475) or VSL#3 thrice weekly via oral gavage. As expected, GC treatment led to significant gut barrier dysfunction (assessed by measuring serum endotoxin levels) and bone loss after 4 weeks. Serum endotoxin levels significantly and negatively correlated with bone volume. Importantly, LR 6475 treatment effectively prevented both GC-induced increase in serum endotoxin and trabecular bone loss. VSL#3 had intermediate results, not differing from either control or GC-treated animals. GC-induced reductions in femur length, cortical thickness, and cortical area were not affected by probiotic treatment. Taken together, these results are the first to demonstrate that LR 6475 effectively prevents the detrimental effects of GC treatment on gut barrier, which correlates with enhanced trabecular bone health in an oral mouse model of GIO. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Post-antibiotic gut dysbiosis-induced trabecular bone loss is dependent on lymphocytes.

Recent studies in mouse models have shown that gut microbiota significantly influences bone health. We demonstrated that 2-week oral treatment with broad spectrum antibiotics followed by 4 weeks of recovery of the gut microbiota results in dysbiosis (microbiota imbalance)-induced bone loss in mice. Because gut microbiota is critical for the development of the immune system and since both microbiota and the immune system can regulate bone health, in this study, we tested the role of the immune system in mediating post-antibiotic dysbiosis-induced bone loss. For this, we treated wild-type (WT) and lymphocyte deficient Rag2 knockout (KO) mice with ampicillin/neomycin cocktail in water for 2 weeks followed by 4 weeks of water without antibiotics. This led to a significant bone loss (31% decrease from control) in WT mice. Interestingly, no bone loss was observed in the KO mice suggesting that lymphocytes are required for dysbiosis-induced bone loss. Bray-Curtis diversity metrics showed similar microbiota changes in both the WT and KO post-antibiotic treated groups. However, several operational taxonomic units (OTUs) classified as Lactobacillales were significantly higher in the repopulated KO when compared to the WT mice, suggesting that these bacteria might play a protective role in preventing bone loss in the KO mice after antibiotic treatment. The effect of dysbiosis on bone was therefore examined in the WT mice in the presence or absence of oral Lactobacillus reuteri treatment for 4 weeks (post-ABX treatment). As hypothesized, mice treated with L. reuteri did not display bone loss, suggesting a bone protective role for this group of bacteria. Taken together, our studies elucidate an important role for lymphocytes in regulating post-antibiotic dysbiosis-induced bone loss.

Involvement of the Gut Microbiota and Barrier Function in Glucocorticoid-Induced Osteoporosis.

Glucocorticoids (GCs) are potent immune-modulating drugs with significant side effects, including glucocorticoid-induced osteoporosis (GIO). GCs directly induce osteoblast and osteocyte apoptosis but also alter intestinal microbiota composition. Although the gut microbiota is known to contribute to the regulation of bone density, its role in GIO has never been examined. To test this, male C57/Bl6J mice were treated for 8 weeks with GC (prednisolone, GC-Tx) in the presence or absence of broad-spectrum antibiotic treatment (ABX) to deplete the microbiota. Long-term ABX prevented GC-Tx-induced trabecular bone loss, showing the requirement of gut microbiota for GIO. Treatment of GC-Tx mice with a probiotic (Lactobacillus reuteri [LR]) prevented trabecular bone loss. Microbiota analyses indicated that GC-Tx changed the abundance of Verrucomicobiales and Bacteriodales phyla and random forest analyses indicated significant differences in abundance of Porphyromonadaceae and Clostridiales operational taxonomic units (OTUs) between groups. Furthermore, transplantation of GC-Tx mouse fecal material into recipient naïve, untreated WT mice caused bone loss, supporting a functional role for microbiota in GIO. We also report that GC caused intestinal barrier breaks, as evidenced by increased serum endotoxin level (2.4-fold), that were prevented by LR and ABX treatments. Enhancement of barrier function with a mucus supplement prevented both GC-Tx-induced barrier leakage and trabecular GIO. In bone, treatment with ABX, LR or a mucus supplement reduced GC-Tx-induced osteoblast and osteocyte apoptosis. GC-Tx suppression of Wnt10b in bone was restored by the LR and high-molecular-weight polymer (MDY) treatments as well as microbiota depletion. Finally, we identified that bone-specific Wnt10b overexpression prevented GIO. Taken together, our data highlight the previously unappreciated involvement of the gut microbiota and intestinal barrier function in trabecular GIO pathogenesis (including Wnt10b suppression and osteoblast and osteocyte apoptosis) and identify the gut as a novel therapeutic target for preventing GIO. © 2019 American Society for Bone and Mineral Research.

Beneficial effects of Lactobacillus reuteri 6475 on bone density in male mice is dependent on lymphocytes.

Oral treatment with probiotic bacteria has been shown to prevent bone loss in multiple models of osteoporosis. In previous studies we demonstrated that oral administration of Lactobacillus reuteri in healthy male mice increases bone density. The host and bacterial mechanisms of these effects however are not well understood. The objective of this study was to understand the role of lymphocytes in mediating the beneficial effects of L. reuteri on bone health in male mice. We administered L. reuteri in drinking water for 4 weeks to wild type or Rag knockout (lack mature T and B lymphocytes) male mice. While L. reuteri treatment increased bone density in wild type, no significant increases were seen in Rag knockout mice, suggesting that lymphocytes are critical for mediating the beneficial effects of L. reuteri on bone density. To understand the effect of L. reuteri on lymphocytes in the intestinal tissues, we isolated mesenteric lymph node (MLN) from naïve wild type mice. In ex vivo studies using whole mesenteric lymph node (MLN) as well as CD3+ T-cells, we demonstrate that live L. reuteri and its secreted factors have concentration-dependent effects on the expression of cytokines, including anti-inflammatory cytokine IL-10. Fractionation studies identified that the active component of L. reuteri is likely water soluble and small in size (<3 kDa) and its effects on lymphocytes are negatively regulated by a RIP2 inhibitor, suggesting a role for NOD signaling. Finally, we show that T-cells from MLNs treated with L. reuteri supernatants, secrete factors that enhance osterix (transcription factor involved in osteoblast differentiation) expression in MC3T3-E1 osteoblasts. Together, these data suggest that L. reuteri secreted factors regulate T-lymphocytes which play an important role in mediating the beneficial effects of L. reuteri on bone density.

Probiotic Lactobacillus reuteri Prevents Postantibiotic Bone Loss by Reducing Intestinal Dysbiosis and Preventing Barrier Disruption.

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 < 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.

Exercise prevents high fat diet-induced bone loss, marrow adiposity and dysbiosis in male mice.

High fat diets can have detrimental effects on the skeleton as well as cause intestinal dysbiosis. Exercise prevents high fat (HF) diet-induced obesity and also improves bone density and prevents the intestinal dysbiosis that promotes energy storage. Previous studies indicate a link between intestinal microbial balance and bone health. Therefore, we examined whether exercise could prevent HF-induced bone pathology in male mice and determined whether benefits correlate to changes in host intestinal microbiota. Male C57Bl/6 mice were fed either a low fat diet (LF; 10 kcal% fat) or a HF diet (60 kcal% fat) and put under sedentary or voluntary exercise conditions for 14 weeks. Our results indicated that HF diet reduced trabecular bone volume, when corrected for differences in body weight, of both the tibia (40% reduction) and vertebrae (25% reduction) as well and increased marrow adiposity (44% increase). More importantly, these effects were prevented by exercise. Exercise also had a significant effect on several cortical bone parameters and enhanced bone mechanical properties in LF but not HF fed mice. Microbiome analyses indicated that exercise altered the HF induced changes in microbial composition by reducing the Firmicutes/Bacteriodetes ratio. This ratio negatively correlated with bone volume as did levels of Clostridia and Lachnospiraceae. In contrast, the abundance of several Actinobacteria phylum members (i.e., Bifidobacteriaceae) were positively correlated with bone volume. Taken together, exercise can prevent many of the negative effects of a high fat diet on male skeletal health. Exercise induced changes in microbiota composition could represent a novel mechanism that contributes to exercise induced benefits to bone health.

Immunology of Gut-Bone Signaling.

In recent years a link between the gastrointestinal tract and bone health has started to gain significant attention. Dysbiosis of the intestinal microbiota has been linked to the pathology of a number of diseases which are associated with bone loss. In addition modulation of the intestinal microbiota with probiotic bacteria has revealed to have both beneficial local and systemic effects. In the present chapter, we discuss the intestinal and bone immune systems, explore how intestinal disease affects the immune system, and examine how these pathologic changes could adversely impact bone health.

Epithelial Barrier Function in Gut-Bone Signaling.

The intestinal epithelial barrier plays an essential role in maintaining host homeostasis. The barrier regulates nutrient absorption as well as prevents the invasion of pathogenic bacteria in the host. It is composed of epithelial cells, tight junctions, and a mucus layer. Several factors, such as cytokines, diet, and diseases, can affect this barrier. These factors have been shown to increase intestinal permeability, inflammation, and translocation of pathogenic bacteria. In addition, dysregulation of the epithelial barrier can result in inflammatory diseases such as inflammatory bowel disease. Our lab and others have also shown that barrier disruption can have systemic effects including bone loss. In this chapter, we will discuss the current literature to understand the link between intestinal barrier and bone. We will discuss how inflammation, aging, dysbiosis, and metabolic diseases can affect intestinal barrier-bone link. In addition, we will highlight the current suggested mechanism between intestinal barrier and bone.

Probiotics in Gut-Bone Signaling.

The intestinal environment is linked to an array of conditions and diseases, including osteoporosis. Human and animal studies indicate that probiotics can benefit intestinal health and may provide a useful therapeutic to prevent and/or treat bone loss. Probiotics are defined as live microorganisms that when administered in adequate amounts will confer a health benefit on the host. In this review, we will focus on (1) probiotics (definition, history, nomenclature, types), (2) the effects of probiotics on bone health, and (3) mechanisms of probiotic prevention of bone pathologies.

The Potential of Probiotics as a Therapy for Osteoporosis.

Osteoporosis, characterized by low bone mass and micro-architectural deterioration of bone tissue with increased risk of fracture, can be categorized into two forms: primary and secondary, depending on whether it occurs as part of the natural aging process (estrogen deficiency) or as part of disease pathology. In both forms bone loss is due to an imbalance in the bone remodeling process, with resorption/formation skewed more toward bone loss. Recent studies and emerging evidence consistently demonstrate the potential of the intestinal microbiota to modulate bone health. This review discusses the process of bone remodeling and the pathology of osteoporosis and introduces the intestinal microbiota and its potential to influence bone health. In particular, we highlight recent murine studies that examine how probiotic supplementation can both increase bone density in healthy individuals and protect against primary (estrogen deficiency) as well as secondary osteoporosis. Potential mechanisms are described to account for how probiotic treatments could be exerting their beneficial effect on bone health.