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.

High Molecular Weight Polymer Promotes Bone Health and Prevents Bone Loss Under Salmonella Challenge in Broiler Chickens.

As a consequence of rapid growth, broiler chickens are more susceptible to infection as well as bone fractures that result in birds being culled. Intestinal infection/inflammation has been demonstrated to promote bone loss in mice and humans. Given this link, we hypothesize that therapeutics that target the gut can benefit bone health. To test this, we infected broiler chickens (7 days old) with Salmonella and treated the birds with or without MDY, a non-absorbable mucus supplement known to benefit intestinal health, from day 1-21 or from day 14-21. Chicken femoral trabecular and cortical bone parameters were analyzed by microcomputed tomography at 21 days. Birds infected with Salmonella displayed significant trabecular bone loss and bone microarchitecture abnormalities that were specific to the femoral neck region, a common site of fracture in chickens. Histological analyses of the chicken bone indicated an increase in osteoclast surface/bone surface in this area indicating that infection-induced bone resorption likely causes the bone loss. Of great interest, treatment with MDY effectively prevented broiler chicken bone loss and architectural changes when given chronically throughout the experiment or for only a week after infection. The latter suggests that MDY may not only prevent bone loss but reverse bone loss. MDY also increased cortical bone mineral density in Salmonella-treated chickens. Taken together, our studies demonstrate that Salmonella-induced bone loss in broiler chickens is prevented by oral MDY.

2,3,7,8-Tetrachlorodibenzo-p-dioxin dose-dependently increases bone mass and decreases marrow adiposity in juvenile mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) agonists have been shown to regulate bone development and remodeling in a species-, ligand-, and age-specific manner, however the underlying mechanisms remain poorly understood. In this study, we characterized the effect of 0.01-30 µg/kg TCDD on the femoral morphology of male and female juvenile mice orally gavaged every 4 days for 28 days and used RNA-Seq to investigate gene expression changes associated with the resultant phenotype. Micro-computed tomography revealed that TCDD dose-dependently increased trabecular bone volume fraction (BVF) 2.9- and 3.3-fold in male and female femurs, respectively. Decreased serum tartrate-resistant acid phosphatase (TRAP) levels, combined with a reduced osteoclast surface to bone surface ratio and repression of femoral proteases (cathepsin K, matrix metallopeptidase 13), suggests that TCDD impaired bone resorption. Increased osteoblast counts at the trabecular bone surface were consistent with a reciprocal reduction in the number of bone marrow adipocytes, suggesting AhR activation may direct mesenchymal stem cell differentiation towards osteoblasts rather than adipocytes. Notably, femoral expression of transmembrane glycoprotein NMB (Gpnmb; osteoactivin), a positive regulator of osteoblast differentiation and mineralization, was dose-dependently induced up to 18.8-fold by TCDD. Moreover, increased serum levels of 1,25-dihydroxyvitamin D3 were in accordance with the renal induction of 1α-hydroxylase Cyp27b1 and may contribute to impaired bone resorption. Collectively, the data suggest AhR activation tipped the bone remodeling balance towards bone formation, resulting in increased bone mass with reduced marrow adiposity.

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.

Estrogen Deficiency Exacerbates Type 1 Diabetes-Induced Bone TNF-α Expression and Osteoporosis in Female Mice.

Estrogen deficiency after menopause is associated with rapid bone loss, osteoporosis, and increased fracture risk. Type 1 diabetes (T1D), characterized by hypoinsulinemia and hyperglycemia, is also associated with bone loss and increased fracture risk. With better treatment options, T1D patients are living longer; therefore, the number of patients having both T1D and estrogen deficiency is increasing. Little is known about the mechanistic impact of T1D in conjunction with estrogen deficiency on bone physiology and density. To investigate this, 11-week-old mice were ovariectomized (OVX), and T1D was induced by multiple low-dose streptozotocin injection. Microcomputed tomographic analysis indicated a marked reduction in trabecular bone volume fraction (BVF) in T1D-OVX mice (~82%) that was far greater than the reductions (~50%) in BVF in either the OVX and T1D groups. Osteoblast markers, number, and activity were significantly decreased in T1D-OVX mice, to a greater extent than either T1D or OVX mice. Correspondingly, marrow adiposity was significantly increased in T1D-OVX mouse bone. Bone expression analyses revealed that tumor necrosis factor (TNF)-α levels were highest in T1D-OVX mice and correlated with bone loss, and osteoblast and osteocyte death. In vitro studies indicate that estrogen deficiency and high glucose enhance TNF-α expression in response to inflammatory signals. Taken together, T1D combined with estrogen deficiency has a major effect on bone inflammation, which contributes to suppressed bone formation and osteoporosis. Understanding the mechanisms/effects of estrogen deficiency in the presence of T1D on bone health is essential for fracture prevention in this patient population.

Intestinal inflammation without weight loss decreases bone density and growth.

Increasing evidence indicates a strong link between intestinal health and bone health. For example, inflammatory bowel disease can cause systemic inflammation, weight loss, and extra-intestinal manifestations, such as decreased bone growth and density. However, the effects of moderate intestinal inflammation without weight loss on bone health have never been directly examined; yet this condition is relevant not only to IBD but to conditions of increased intestinal permeability and inflammation, as seen with ingestion of high-fat diets, intestinal dysbiosis, irritable bowel syndrome, metabolic syndrome, and food allergies. Here, we induced moderate intestinal inflammation without weight loss in young male mice by treating with a low dose of dextran sodium sulfate (1%) for 15 days. The mice displayed systemic changes marked by significant bone loss and a redistribution of fat from subcutaneous to visceral fat pad stores. Bone loss was caused by reduced osteoblast activity, characterized by decreased expression of osteoblast markers (runx2, osteocalcin), histomorphometry, and dynamic measures of bone formation. In addition, we observed a reduction in growth plate thickness and hypertrophic chondrocyte matrix components (collagen X). Correlation analyses indicate a link between gut inflammation and disease score, but more importantly, we observed that bone density measures negatively correlated with intestinal disease score, as well as colon and bone TNF-α levels. These studies demonstrate that colitis-induced bone loss is not dependent upon weight loss and support a role for inflammation in the link between gut and bone health, an important area for future therapeutic development.

Understanding the skeletal pathology of type 1 and 2 diabetes mellitus.

Diabetes affects over 25 million people and is characterized by hyperglycemia resulting from a lack of insulin or reduced insulin sensitivity. A serious complication of diabetes is the increase in fracture risk observed in both type 1 and type 2 diabetic patients. This review focuses on some of the cellular and mechanistic causes of diabetes-induced fracture risk. Type 1 and type 2 diabetes most likely have unique and overlapping mechanisms of bone loss. While type 1 diabetes is associated with reduced bone mineral density, this is not usually seen in type 2 diabetes. Hyperglycemia, present in both type 1 and 2 diabetes, alters bone matrix proteins such as collagen I through nonenzymatic glycation, which can decrease bone toughness and increase fracture risk even in the absence of bone loss. Diabetes is also associated with increased inflammation and altered adipokine and calcitrophic hormone levels, which further contribute to bone pathophysiology. As medical advances significantly lengthen patient lifespan, exposure to diabetic conditions increases and correspondingly so do disease complications. Further research to identify molecular pathways in diabetes-associated bone pathology will provide the basis for therapeutic targets/directions to increase treatment options and improve patient health and well-being.