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Effects of the gut microbiota on bone mass Claes Ohlsson and Klara Sjo¨gren Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, 413 45 Gothenburg, Sweden
The gut microbiota (GM), the commensal bacteria living in our intestine, performs numerous useful functions, including modulating host metabolism and immune status. Recent studies demonstrate that the GM is also a regulator of bone mass and it is proposed that the effect of the GM on bone mass is mediated via effects on the immune system, which in turn regulates osteoclastogenesis. Under normal conditions, the skeleton is constantly remodeled by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCLs), and imbalances in this process may lead to osteoporosis. Here we review current knowledge on the possible role for the GM in the regulation of bone metabolism and propose that the GM might be a novel therapeutic target for osteoporosis and fracture prevention. Osteoporosis, a disease of fragile bones caused by hereditary and environmental factors Fractures caused by osteoporosis constitute a major health concern and result in a huge economic burden on health care systems. The lifetime risk of any osteoporotic fracture is high in the western world (around 50% for women and 20% for men), and fractures are associated with significant mortality and morbidity in the elderly [1]. Osteoporosis is characterized by enhanced skeletal fragility due to reduction in bone quantity and/or quality. Bone strength cannot be directly measured in vivo, but bone mineral density (BMD; see Glossary) is highly correlated with bone strength and is commonly used in the clinic to predict fracture risk [2]. The risk of osteoporosis depends both on how much bone is acquired during skeletal growth and development until peak bone mass is reached at 20–30 years of age, and on the rate of the subsequent age-dependent bone loss. Twin and family studies have shown that between 50% and 85% of the variance in peak bone mass is genetically determined, and that there is a heritable component also for age-related bone loss, but environmental factors seem to play a relatively more pronounced role in this latter process [3]. In recent years, the importance of the GM for both health and disease has been intensively studied. The GM constitutes trillions of bacteria, which collectively Corresponding author: Sjo¨gren, K. ([email protected]). Keywords: osteoporosis; osteoimmunology; gut microbiota. 1043-2760/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.11.004
contain 150-fold more genes than our human genome. It is acquired at birth and, although a distinct entity, it has clearly coevolved with the human genome and can be considered a multicellular organ that communicates with and affects its host in numerous ways [4]. The composition of the GM is modulated by a number of environmental factors such as diet and antibiotic treatments [5–7]. Molecules produced by the gut bacteria can be both beneficial and harmful and are known to affect endocrine cells in the gut, the enteric nervous system, gut permeability, and the immune system. At homeostasis, the GM provides colonization resistance with epithelial and immune balance, protecting the host from invading bacteria, viruses, and possibly other classes of pathogens. Perturbations in this balance can be caused by pathogens, antibiotic treatment, and diet causing inflammation, tissue destruction, and dysbiosis that may lead to disease development [8]. Perturbed microbial composition has been postulated to be involved in a range of inflammatory conditions, within and outside the gut, including inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, diabetes, food allergies, eczema, and asthma, as well as obesity and the metabolic syndrome [9,10]. The inflammation seen
Glossary Bone mineral density (BMD): is highly correlated with bone strength and is commonly measured and used in the clinic to predict fracture risk. According to the World Health Organization’s guidelines, osteoporosis is diagnosed when the BMD is more than or equal to 2.5 standard deviations below that of a young adult reference population. Dysbiosis: refers to an imbalance between putative species of ‘protective’ versus ‘harmful’ intestinal bacteria. Gnotobiotic: is an animal that is free of bacteria or contaminants or into which a known microorganism or contaminant has been introduced for research purposes. Inulins: are plant-derived polysaccharides. The inulins belong to a class of dietary fibers known as fructans. Microbiota: refers to the microflora and microfauna in an ecosystem. Osteoblasts (OBs): are cells that make bone by producing a matrix that then becomes mineralized. The skeleton is preserved by a balance between the activity of OBs that form bone and OCLs that break it down. Osteoclasts (OCLs): are cells that break down bone and are responsible for bone resorption. OCLs are large multinucleate cells that differentiate from hematopoietic stem cells in the bone marrow. Prebiotics: are non-digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and act as a substrate for commensal bacteria. They help promote the growth and activity of advantageous bacteria in the gut. Probiotics: the Food and Agriculture Organization of the United Nations and the WHO (FAO/WHO) definition of probiotics is ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Probiotics such as lactobacilli and bifidobacteria are beneficial to the host because they help improve the intestinal bacterial balance.
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Review in inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis is associated with alterations in the diversity and richness of the GM [11–13]. A study in monozygotic twins, discordant or concordant for Crohn’s disease, showed an increased ratio of adherent invasive Escherichia coli to Faecalibacterium prausnitzii in diseased twins [14]. F. prausnitzii has anti-inflammatory functions indicating that the ratio of pathogenic to more protective species is of importance [15]. In a large multicenter analysis of fecal microbiota profiles in patients with Crohn’s disease, a fecal microbiota profile predicting disease activity was identified. The genus Bifidobacterium was significantly decreased during the active phase of Crohn’s disease and increased to healthy levels during the remission phase [16]. Species of the Bifidobacterium genus are normal inhabitants of a healthy human gut and alterations in intestinal bifidobacteria levels, or species composition, are often present in cases of GM dysbiosis [17]. However, despite the large number of studies linking alterations in the GM to inflammatory diseases, it is unclear whether these alterations are the cause or consequence of these diseases. Gut-associated inflammatory and autoimmune conditions have been associated with low bone mass, suggesting a connection between the gut and bone [18,19]. The GM affects the host’s immune status and it is well established that there is a connection between the immune system and bone metabolism, suggesting that the GM might affect bone metabolism via altered immune status. Here we review current knowledge supporting a role for the GM in the regulation of bone metabolism. Bone metabolism – the immune connection The skeleton is remodeled by bone-forming OBs and boneresorbing OCLs [20]. Parathyroid hormone, vitamin D, retinoids, thyroid hormones, cortisol, inflammatory cytokines, and sex-steroids are all important regulators of skeletal remodeling and thereby the risk of osteoporosis. Besides providing structural support, the skeleton also serves as a niche for mesenchymal and hematopoietic progenitors. OBs are derived from pluripotent mesenchymal stromal cells while OCLs are derived from hematopoietic stem cells that also generate immune cells [21]. OCLs are specifically derived from the myeloid-monocyte lineage of hematopoietic cells and it is the local microenvironment that determines whether the myeloid precursor cell will differentiate into a macrophage, a myeloid dendritic cell, or an OCL. The presence of macrophage colony stimulating factor (M-CSF) leads to increased proliferation and survival as well as upregulated expression of receptor activator of nuclear factor-kB (RANK) in OCL precursor cells. This allows RANK ligand (RANKL) to bind and start the signaling cascade that leads to OCL formation [21]. The association between inflammation and bone loss is well established and in auto-immune diseases such as rheumatoid arthritis, osteoclastic bone resorption is driven by inflammatory cytokines produced by activated T cells [22]. The estrogen deficiency that occurs at menopause results in increased formation and prolonged survival of OCLs. This is suggested to be due to a number of factors including loss of the immunosuppressive effects of estrogen, resulting in increased production of cytokines 2
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promoting osteoclastogenesis, and direct effects of estrogen on OCLs [23,24]. Several studies indicate that low-grade inflammation affects physiological bone turnover and plays a role in pathological skeletal conditions such as osteoporosis. Moderately elevated serum levels of high sensitivity C-reactive protein (hsCRP), as an estimate of low-grade systemic inflammation, are reported to be associated with low BMD, elevated bone resorption, bone loss, and increased fracture risk [25–28]. In line with these data, blockade of the inflammatory cytokines tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-1) leads to a decrease in bone resorption markers in early postmenopausal women [29]. In addition, mice depleted of T cells in vivo by treatment with anti-CD4 and anti-CD8 antibodies are protected against ovariectomy (ovx)-induced bone loss [30]. The mechanism involves an upregulation of TNFproducing T cells in the bone marrow of ovx mice, further arguing for a role of T cells and T cell-produced cytokines in bone turnover [31]. To summarize, imbalances in bone remodeling may lead to bone loss and osteoporosis. The GM as a regulator of bone mass At birth, we are immediately colonized with bacteria from our mother and the environment. The GM is varied at first but stabilizes towards an adult-like configuration during the 3-year period after birth [32,33]. During this time, the neonatal immune system rapidly matures under the influence of the GM and environmental factors such as diet, intestinal infections, antibiotic treatments, and breastfeeding. The GM composition changes with age and is extremely variable between older individuals (>65 years) [34–36]. The GM presents a vast source of potential antigens for the host’s immune system to cope with. At homeostasis, there is a symbiotic relationship between the host and resident microbes that helps in food digestion and protects from invading pathogens. However, under pathological conditions that compromise the host’s ability to limit the microbiota’s entry from the intestines, species can invade host tissue and cause disease. Altered immune stimulation or release of metabolic products by the GM can also result in disease. Dietary changes, antibiotic treatments, or pathogens can shift the composition of the GM, and thereby disturb the balance in metabolic and immune regulatory networks that normally restrain intestinal inflammation [9,10,37,38]. The use of gnotobiotic animals and the development of effective sequencing technologies have made it possible to characterize the effects and the composition of the GM. Studies have shown that germ-free (GF) animals have immature mucosal immune systems with poorly developed gut-associated lymphoid tissue (GALT). Furthermore, GF mice have a reduced number of CD4+ T cells in the spleen and fewer and smaller germinal centers within the spleen, suggesting that the GM is capable of shaping systemic immunity [39]. It was recently shown that absence of GM in GF mice leads to increased bone mass, compared to conventionally raised (CONV-R) mice [40,41]. It was found that both the spongy trabecular bone and the compact cortical bone was affected with the trabecular bone volume/tissue volume (BV/TV) increased by 39% in the distal femur of GF compared with CONV-R mice [40]. The increased BV/TV was
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Review associated with increased trabecular number and decreased trabecular separation, whereas trabecular thickness was unchanged in GF compared with CONV-R mice. In addition, cortical bone area was also increased in GF mice. A decrease in the number of OCLs was present in bone and in bone marrow cultures from GF mice, and bone marrow exhibited decreased frequency of CD4+ T cells and OCL precursor cells (CD11b+/Gr1 ). By contrast, the bone formation rate was not significantly altered in the GF mice, suggesting that the increased bone mass in the GF mice is predominantly caused by reduced bone resorption as a result of inhibited osteoclastogenesis. As the GF mice also had reduced mRNA levels of the osteolytic cytokines IL-6 and TNFa in the bone, it can be suggested that the decreased osteoclastogenesis was caused by immunemediated mechanisms. Importantly, colonization of mice born in a germ-free environment with GM from mice raised in a conventional environment at 3 weeks of age led to a normalization of both trabecular and cortical bone mass and the frequency of CD4+ T cells and OCL precursor cells in bone marrow. It was proposed that the increased bone mass in GF mice is caused by fewer CD4+ cells recirculating in the blood and secondary lymphoid tissue, resulting in a decreased frequency of CD4+ T cells in bone marrow. The increased bone mass was associated with a reduction in the expression of inflammatory cytokines in bone and less osteoclastogenesis [40]. It cannot be excluded that other mechanisms might also contribute to the high bone mass phenotype in GF mice. However, it is unlikely that the increased bone mass is caused by an altered calcium metabolism, because serum calcium and hormones regulating calcium homeostasis are normal in GF mice [40]. It has been reported that gutderived circulating serotonin inhibits bone formation and reduces bone mass [42]. Indeed, serum serotonin levels were decreased in GF mice, however, colonization of GF mice with GM from mice raised in a conventional environment, which leads to the normalization of bone mass, did not significantly affect serum serotonin levels, indicating that the high bone mass in GF mice may not be a direct effect of altered serum serotonin levels [40]. We propose that, thus far, the most probable mechanism by which GM affects bone mass involves altered systemic and bone marrow immune status, which in turn regulates osteoclastogenesis (Figure 1). GM, sex hormones, and bone loss In a recent study, Li et al. investigated the role of the GM in the bone loss induced by sex-steroid deficiency [41]. GF and CONV-R mice were treated with vehicle or Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist that blocks sex-steroid production, thus mimicking ovx. Interestingly, Leuprolide caused a significantly greater cortical and trabecular bone loss in CONV-R than in GF mice, demonstrating that the GM plays a pivotal role in the bone loss induced by sex-steroid deficiency. Serum levels of C-terminal telopeptide (CTX), a marker of bone resorption, were increased by Leuprolide in CONV-R but not in GF mice, suggesting that the GF mice are protected from the enhanced bone resorption normally occurring
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Gut microbiota Probiocs
Diet
Prebiocs
Anbiocs
? Calcium metabolism
? Immune system
Gut-de rived Gut-derived serotonin
TRENDS in Endocrinology & Metabolism
Figure 1. The gut microbiota (GM) as a proposed regulator of bone mass. Recent studies using GF mice demonstrate that the GM is a regulator of bone mass, and we propose that the inhibitory effect of the GM on bone mass is mainly mediated via effects on the immune system, which in turn regulates osteoclastogenesis. However, we cannot fully exclude that other mechanisms such as altered calcium metabolism and regulation of gut-derived serotonin also contribute. Diet is an environmental factor affecting the composition of the GM. Studies have shown that antibiotic, probiotic, and prebiotic treatments, which have an impact on GM composition, regulate bone metabolism. We propose that the GM might be a promising novel therapeutic target for osteoporosis.
in sex-steroid-deficient mice. Moreover, Leuprolide increased the frequency of TNFa+CD4+ and TNFa+CD8+ T cells in the bone marrow of CONV-R mice, but not of GF mice. These findings suggest that the GM is required to shape the immune system to respond to sex-steroid deficiency. The authors conclude that the GM plays a significant role in inducing bone loss and increasing bone turnover in sex-steroid-deficient mice by providing the antigens required for bone marrow T cell expansion and increased TNFa production [41]. Furthermore, they propose that the GM composition may be involved in the regulation of the magnitude of bone loss experienced by postmenopausal women. One may speculate that an increased inflammatory status caused by an unfavorable GM composition would result in a greater bone loss when women lose the immunosuppressive effects of estrogen after menopause. GM composition and bone loss The GM composition can be modulated by environmental factors, such as diet and treatments with antibiotics, prebiotics, and probiotics (Figure 1). A role of the GM in bone mass regulation is supported by a study demonstrating that subtherapeutic antibiotic treatment to mice at weaning increases bone mass after 3 weeks of exposure [37]. Although the low dose of antibiotics in this study did not cause a significant alteration in bacterial count, it caused shifts in the composition of the GM. In a followup study, low-dose penicillin delivered from birth or from weaning caused an increased BMD in adult female mice [38]. Furthermore, tetracycline treatment has been shown to prevent bone loss after ovx, partly due to inhibited bone resorption [43], and in a similar manner as described for the sex-steroid-deficient GF mice [41]. In another study, tetracycline treatment improved mechanical 3
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Review properties of bone after ovx [44]. These studies demonstrate that antibiotic treatment has the capacity to influence both the GM composition and bone mass, supporting the notion that the GM is a regulator of bone homeostasis. Probiotics and prebiotics Low-dose antibiotic treatment has been used as a growthpromoting agent in farm animals for decades [45]. Because of residuals in meat products and the development of antibiotic-resistant bacteria, this practice is now banned in Europe. This has raised the interest in using other treatments such as probiotics and prebiotics to improve health status and growth. Probiotics are defined as live commensal microorganisms such as bacteria or fungi that when administered in adequate amounts can confer a health benefit on the host such as improved intestinal function and integrity of the intestinal lining. They may also positively affect immune responses in the host gastrointestinal tract to promote health. Several probiotic strains of commensal bacteria exist that are used in experimental settings and some are currently being examined to treat inflammatory bowel disease [46]. Prebiotics are non-digestible food ingredients such as oligosaccharides and dietary fiber that stimulate the growth and/or activity of beneficial bacteria in the digestive system in ways claimed to be beneficial to health [47]. The suggested underlying mechanisms conferring these beneficial effects are manifold and include increased solubility and absorption of minerals as well as anti-inflammatory effects [48]. The effect of probiotics on bone mass has been evaluated in chickens treated during 6 weeks with a diet supplemented with or without two probiotic strains, demonstrating that the probiotic supplementation increased bone mass in the tibia [49]. As several studies indicate that the GM modulates the magnitude of the bone loss in sex-steroid-deficient female mice, it was hypothesized that treatment with probiotics might protect mice from ovx-induced bone loss [41,43,44]. A single (Lactobacillus paracasei DSM13434; L. para) or a mixture (L. paracasei DSM13434, Lactobacillus plantarum DSM 15312, and L. plantarum DSM 15313; L. mix) of probiotic strains was selected based on their anti-inflammatory properties, as shown in an earlier study [50], and was given in the drinking water during a 6-week period to mice, initiated prior to ovx. Both the L. para and the L. mix treatments were shown to protect the mice from ovx-induced bone loss and bone resorption [51]. Subsequent mechanistic studies revealed that the probiotic treatment reduced the expression of two inflammatory cytokines, TNFa and IL-1b, and increased the expression of osteoprotegerin (OPG), a potent inhibitor of osteoclastogenesis, in bone of ovx mice. A bone-protective effect of probiotic treatment using a different strain in ovx mice has also been reported. Britton et al. tested the effect of Lactobacillus reuteri, a commensal bacterium that also secretes beneficial immunomodulatory factors, on ovx-related bone loss. Treatment with L. reuteri altered the GM composition and prevented ovx-induced trabecular bone loss and bone resorption [52]. In addition, this treatment suppressed the ovx-induced increase in bone marrow CD4+ T cells, supporting the notion that the GM modulates the 4
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immune status in bone, and thereby affects OCL-mediated bone resorption. In a separate study, oral administration of L. reuteri treatment was shown to decrease intestinal inflammation and increase trabecular bone mass in gonadal intact male mice [53]. Taken together, these studies suggest that treatment with probiotics has beneficial effects on the immune status in bone, which in turn results in attenuated bone resorption in both ovx female mice and intact male mice. The effect of the prebiotic galactooligosaccharide (GOS) on GM composition and bone mass has been evaluated in growing rats and humans. In rats, GOS supplementation altered the GM composition, with an increase in the relative proportion of bifidobacteria, and increased the bone mass [54]. In a recent study, GOS supplementation was given to adolescent girls during a 3-week period to test the dose–response effect on calcium absorption [55]. GOS treatment improved calcium absorption and increased the relative proportion of bifidobacteria in the GM similarly to what was earlier observed in growing rats. In addition, Abrams et al. showed that prebiotic treatment enhanced bone mineralization during pubertal growth [56]. The prebiotic used in that study was a mixture of short- and long-chain inulin-type fructans given as a daily food supplement for a period of 1 year, and resulted in a 47% greater increase in BMD compared to the control group. The authors proposed that this effect on BMD was mainly due to improved calcium absorption but the GM composition and possible systemic immune-mediated effects were not evaluated. Collectively, the studies using antibiotic treatment, probiotics, or prebiotics indicate that treatments affecting the GM composition also regulate bone metabolism. One possible mechanism, as suggested by the findings from GF mice, is that the GM exerts its effects on bone mass by influencing the host’s immune system. Concluding remarks and future perspectives In summary, the GM modulates host metabolism and immune status. Recent studies using GF mice demonstrate that the GM is a regulator also of bone mass and it is proposed that the inhibitory effect of the GM on bone mass is mediated via effects on immune status, which in turn regulates osteoclastogenesis. A role of the GM in bone metabolism is further supported by studies demonstrating that antibiotic, probiotic, and prebiotic treatments that impact GM composition regulate bone metabolism. The effect of these treatments is present in periods of rapid growth, such as puberty, when bone mass acquisition is large and during periods of bone loss, such as castration. Recent studies show that the bone loss caused by sexsteroid deficiency is diminished in GF mice and can be prevented by treatment with probiotics. Collectively, these studies suggest that the GM may be a novel therapeutic target for osteoporosis (Figure 1). Treatment with probiotics has already been shown to improve bone mass in rodent models of bone loss, but future randomized clinical trials are required to determine the possible effect of probiotics and other novel therapies modulating the GM composition on bone mass and fracture risk in patients with osteoporosis.
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Review The GF mouse is a useful model to study the role of the GM in bone mass regulation. Genetically engineered GF mouse models, in which selected genes are deleted or overexpressed, can help to identify the molecular mechanism by which the GM affects bone mass. Access to cheaper sequencing and improved bioinformatics tools will allow metagenomic sequencing for the analysis of the GM composition in large prospective cohort studies. This can be used to evaluate the predictive value of the GM composition as a biomarker for low bone mass and fracture risk. In addition, metatranscriptomics and metaproteomics will most likely be used to identify the microbial genes and proteins that have an impact on bone mass and fracture risk. We propose a new cross-disciplinary GM–bone research field called ‘osteo-microbiology’, bridging the gaps between bone physiology, gastroenterology, immunology, and microbiology. Future studies are clearly warranted in this research field to determine if the GM composition might be used as a biomarker for fracture risk prediction and to validate the GM as a possible novel therapeutic target for osteoporosis. Acknowledgments This work was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, COMBINE, the Avtal om La¨karutbildning och Forskning/La¨karutbildningsavtalet research grant in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar So¨derberg Foundation, the Novo Nordisk Foundation, the Magnus Bergvall ˚ ke Wiberg Foundation. Foundation, and the A
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50 Lavasani, S. et al. (2010) A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL10 producing regulatory T cells. PLoS ONE, Published online February 2, 2010, http://dx.doi.org/10.1371/journal.pone.0009009 51 Ohlsson, C. et al. (2014) Probiotics protect mice from ovariectomyinduced cortical bone loss. PLoS ONE, Published online March 17, 2014, http://dx.doi.org/10.1371/journal.pone.0092368 52 Britton, R.A. et al. (2014) Probiotic L. reuteri treatment prevents bone loss in a menopausal ovariectomized mouse model. J. Cell. Physiol. 229, 1822–1830 53 McCabe, L.R. et al. (2013) Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice. J. Cell. Physiol. 228, 1793–1798 54 Weaver, C.M. et al. (2011) Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. J. Agric. Food Chem. 59, 6501–6510 55 Whisner, C.M. et al. (2013) Galacto-oligosaccharides increase calcium absorption and gut bifidobacteria in young girls: a double-blind crossover trial. Br. J. Nutr. 110, 1292–1303 56 Abrams, S.A. et al. (2005) A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am. J. Clin. Nutr. 82, 471–476
Review
Effects of the gut microbiota on bone mass Claes Ohlsson and Klara Sjo¨gren Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, 413 45 Gothenburg, Sweden
The gut microbiota (GM), the commensal bacteria living in our intestine, performs numerous useful functions, including modulating host metabolism and immune status. Recent studies demonstrate that the GM is also a regulator of bone mass and it is proposed that the effect of the GM on bone mass is mediated via effects on the immune system, which in turn regulates osteoclastogenesis. Under normal conditions, the skeleton is constantly remodeled by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCLs), and imbalances in this process may lead to osteoporosis. Here we review current knowledge on the possible role for the GM in the regulation of bone metabolism and propose that the GM might be a novel therapeutic target for osteoporosis and fracture prevention. Osteoporosis, a disease of fragile bones caused by hereditary and environmental factors Fractures caused by osteoporosis constitute a major health concern and result in a huge economic burden on health care systems. The lifetime risk of any osteoporotic fracture is high in the western world (around 50% for women and 20% for men), and fractures are associated with significant mortality and morbidity in the elderly [1]. Osteoporosis is characterized by enhanced skeletal fragility due to reduction in bone quantity and/or quality. Bone strength cannot be directly measured in vivo, but bone mineral density (BMD; see Glossary) is highly correlated with bone strength and is commonly used in the clinic to predict fracture risk [2]. The risk of osteoporosis depends both on how much bone is acquired during skeletal growth and development until peak bone mass is reached at 20–30 years of age, and on the rate of the subsequent age-dependent bone loss. Twin and family studies have shown that between 50% and 85% of the variance in peak bone mass is genetically determined, and that there is a heritable component also for age-related bone loss, but environmental factors seem to play a relatively more pronounced role in this latter process [3]. In recent years, the importance of the GM for both health and disease has been intensively studied. The GM constitutes trillions of bacteria, which collectively Corresponding author: Sjo¨gren, K. ([email protected]). Keywords: osteoporosis; osteoimmunology; gut microbiota. 1043-2760/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.11.004
contain 150-fold more genes than our human genome. It is acquired at birth and, although a distinct entity, it has clearly coevolved with the human genome and can be considered a multicellular organ that communicates with and affects its host in numerous ways [4]. The composition of the GM is modulated by a number of environmental factors such as diet and antibiotic treatments [5–7]. Molecules produced by the gut bacteria can be both beneficial and harmful and are known to affect endocrine cells in the gut, the enteric nervous system, gut permeability, and the immune system. At homeostasis, the GM provides colonization resistance with epithelial and immune balance, protecting the host from invading bacteria, viruses, and possibly other classes of pathogens. Perturbations in this balance can be caused by pathogens, antibiotic treatment, and diet causing inflammation, tissue destruction, and dysbiosis that may lead to disease development [8]. Perturbed microbial composition has been postulated to be involved in a range of inflammatory conditions, within and outside the gut, including inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, diabetes, food allergies, eczema, and asthma, as well as obesity and the metabolic syndrome [9,10]. The inflammation seen
Glossary Bone mineral density (BMD): is highly correlated with bone strength and is commonly measured and used in the clinic to predict fracture risk. According to the World Health Organization’s guidelines, osteoporosis is diagnosed when the BMD is more than or equal to 2.5 standard deviations below that of a young adult reference population. Dysbiosis: refers to an imbalance between putative species of ‘protective’ versus ‘harmful’ intestinal bacteria. Gnotobiotic: is an animal that is free of bacteria or contaminants or into which a known microorganism or contaminant has been introduced for research purposes. Inulins: are plant-derived polysaccharides. The inulins belong to a class of dietary fibers known as fructans. Microbiota: refers to the microflora and microfauna in an ecosystem. Osteoblasts (OBs): are cells that make bone by producing a matrix that then becomes mineralized. The skeleton is preserved by a balance between the activity of OBs that form bone and OCLs that break it down. Osteoclasts (OCLs): are cells that break down bone and are responsible for bone resorption. OCLs are large multinucleate cells that differentiate from hematopoietic stem cells in the bone marrow. Prebiotics: are non-digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and act as a substrate for commensal bacteria. They help promote the growth and activity of advantageous bacteria in the gut. Probiotics: the Food and Agriculture Organization of the United Nations and the WHO (FAO/WHO) definition of probiotics is ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Probiotics such as lactobacilli and bifidobacteria are beneficial to the host because they help improve the intestinal bacterial balance.
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Review in inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis is associated with alterations in the diversity and richness of the GM [11–13]. A study in monozygotic twins, discordant or concordant for Crohn’s disease, showed an increased ratio of adherent invasive Escherichia coli to Faecalibacterium prausnitzii in diseased twins [14]. F. prausnitzii has anti-inflammatory functions indicating that the ratio of pathogenic to more protective species is of importance [15]. In a large multicenter analysis of fecal microbiota profiles in patients with Crohn’s disease, a fecal microbiota profile predicting disease activity was identified. The genus Bifidobacterium was significantly decreased during the active phase of Crohn’s disease and increased to healthy levels during the remission phase [16]. Species of the Bifidobacterium genus are normal inhabitants of a healthy human gut and alterations in intestinal bifidobacteria levels, or species composition, are often present in cases of GM dysbiosis [17]. However, despite the large number of studies linking alterations in the GM to inflammatory diseases, it is unclear whether these alterations are the cause or consequence of these diseases. Gut-associated inflammatory and autoimmune conditions have been associated with low bone mass, suggesting a connection between the gut and bone [18,19]. The GM affects the host’s immune status and it is well established that there is a connection between the immune system and bone metabolism, suggesting that the GM might affect bone metabolism via altered immune status. Here we review current knowledge supporting a role for the GM in the regulation of bone metabolism. Bone metabolism – the immune connection The skeleton is remodeled by bone-forming OBs and boneresorbing OCLs [20]. Parathyroid hormone, vitamin D, retinoids, thyroid hormones, cortisol, inflammatory cytokines, and sex-steroids are all important regulators of skeletal remodeling and thereby the risk of osteoporosis. Besides providing structural support, the skeleton also serves as a niche for mesenchymal and hematopoietic progenitors. OBs are derived from pluripotent mesenchymal stromal cells while OCLs are derived from hematopoietic stem cells that also generate immune cells [21]. OCLs are specifically derived from the myeloid-monocyte lineage of hematopoietic cells and it is the local microenvironment that determines whether the myeloid precursor cell will differentiate into a macrophage, a myeloid dendritic cell, or an OCL. The presence of macrophage colony stimulating factor (M-CSF) leads to increased proliferation and survival as well as upregulated expression of receptor activator of nuclear factor-kB (RANK) in OCL precursor cells. This allows RANK ligand (RANKL) to bind and start the signaling cascade that leads to OCL formation [21]. The association between inflammation and bone loss is well established and in auto-immune diseases such as rheumatoid arthritis, osteoclastic bone resorption is driven by inflammatory cytokines produced by activated T cells [22]. The estrogen deficiency that occurs at menopause results in increased formation and prolonged survival of OCLs. This is suggested to be due to a number of factors including loss of the immunosuppressive effects of estrogen, resulting in increased production of cytokines 2
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promoting osteoclastogenesis, and direct effects of estrogen on OCLs [23,24]. Several studies indicate that low-grade inflammation affects physiological bone turnover and plays a role in pathological skeletal conditions such as osteoporosis. Moderately elevated serum levels of high sensitivity C-reactive protein (hsCRP), as an estimate of low-grade systemic inflammation, are reported to be associated with low BMD, elevated bone resorption, bone loss, and increased fracture risk [25–28]. In line with these data, blockade of the inflammatory cytokines tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-1) leads to a decrease in bone resorption markers in early postmenopausal women [29]. In addition, mice depleted of T cells in vivo by treatment with anti-CD4 and anti-CD8 antibodies are protected against ovariectomy (ovx)-induced bone loss [30]. The mechanism involves an upregulation of TNFproducing T cells in the bone marrow of ovx mice, further arguing for a role of T cells and T cell-produced cytokines in bone turnover [31]. To summarize, imbalances in bone remodeling may lead to bone loss and osteoporosis. The GM as a regulator of bone mass At birth, we are immediately colonized with bacteria from our mother and the environment. The GM is varied at first but stabilizes towards an adult-like configuration during the 3-year period after birth [32,33]. During this time, the neonatal immune system rapidly matures under the influence of the GM and environmental factors such as diet, intestinal infections, antibiotic treatments, and breastfeeding. The GM composition changes with age and is extremely variable between older individuals (>65 years) [34–36]. The GM presents a vast source of potential antigens for the host’s immune system to cope with. At homeostasis, there is a symbiotic relationship between the host and resident microbes that helps in food digestion and protects from invading pathogens. However, under pathological conditions that compromise the host’s ability to limit the microbiota’s entry from the intestines, species can invade host tissue and cause disease. Altered immune stimulation or release of metabolic products by the GM can also result in disease. Dietary changes, antibiotic treatments, or pathogens can shift the composition of the GM, and thereby disturb the balance in metabolic and immune regulatory networks that normally restrain intestinal inflammation [9,10,37,38]. The use of gnotobiotic animals and the development of effective sequencing technologies have made it possible to characterize the effects and the composition of the GM. Studies have shown that germ-free (GF) animals have immature mucosal immune systems with poorly developed gut-associated lymphoid tissue (GALT). Furthermore, GF mice have a reduced number of CD4+ T cells in the spleen and fewer and smaller germinal centers within the spleen, suggesting that the GM is capable of shaping systemic immunity [39]. It was recently shown that absence of GM in GF mice leads to increased bone mass, compared to conventionally raised (CONV-R) mice [40,41]. It was found that both the spongy trabecular bone and the compact cortical bone was affected with the trabecular bone volume/tissue volume (BV/TV) increased by 39% in the distal femur of GF compared with CONV-R mice [40]. The increased BV/TV was
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Review associated with increased trabecular number and decreased trabecular separation, whereas trabecular thickness was unchanged in GF compared with CONV-R mice. In addition, cortical bone area was also increased in GF mice. A decrease in the number of OCLs was present in bone and in bone marrow cultures from GF mice, and bone marrow exhibited decreased frequency of CD4+ T cells and OCL precursor cells (CD11b+/Gr1 ). By contrast, the bone formation rate was not significantly altered in the GF mice, suggesting that the increased bone mass in the GF mice is predominantly caused by reduced bone resorption as a result of inhibited osteoclastogenesis. As the GF mice also had reduced mRNA levels of the osteolytic cytokines IL-6 and TNFa in the bone, it can be suggested that the decreased osteoclastogenesis was caused by immunemediated mechanisms. Importantly, colonization of mice born in a germ-free environment with GM from mice raised in a conventional environment at 3 weeks of age led to a normalization of both trabecular and cortical bone mass and the frequency of CD4+ T cells and OCL precursor cells in bone marrow. It was proposed that the increased bone mass in GF mice is caused by fewer CD4+ cells recirculating in the blood and secondary lymphoid tissue, resulting in a decreased frequency of CD4+ T cells in bone marrow. The increased bone mass was associated with a reduction in the expression of inflammatory cytokines in bone and less osteoclastogenesis [40]. It cannot be excluded that other mechanisms might also contribute to the high bone mass phenotype in GF mice. However, it is unlikely that the increased bone mass is caused by an altered calcium metabolism, because serum calcium and hormones regulating calcium homeostasis are normal in GF mice [40]. It has been reported that gutderived circulating serotonin inhibits bone formation and reduces bone mass [42]. Indeed, serum serotonin levels were decreased in GF mice, however, colonization of GF mice with GM from mice raised in a conventional environment, which leads to the normalization of bone mass, did not significantly affect serum serotonin levels, indicating that the high bone mass in GF mice may not be a direct effect of altered serum serotonin levels [40]. We propose that, thus far, the most probable mechanism by which GM affects bone mass involves altered systemic and bone marrow immune status, which in turn regulates osteoclastogenesis (Figure 1). GM, sex hormones, and bone loss In a recent study, Li et al. investigated the role of the GM in the bone loss induced by sex-steroid deficiency [41]. GF and CONV-R mice were treated with vehicle or Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist that blocks sex-steroid production, thus mimicking ovx. Interestingly, Leuprolide caused a significantly greater cortical and trabecular bone loss in CONV-R than in GF mice, demonstrating that the GM plays a pivotal role in the bone loss induced by sex-steroid deficiency. Serum levels of C-terminal telopeptide (CTX), a marker of bone resorption, were increased by Leuprolide in CONV-R but not in GF mice, suggesting that the GF mice are protected from the enhanced bone resorption normally occurring
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Gut microbiota Probiocs
Diet
Prebiocs
Anbiocs
? Calcium metabolism
? Immune system
Gut-de rived Gut-derived serotonin
TRENDS in Endocrinology & Metabolism
Figure 1. The gut microbiota (GM) as a proposed regulator of bone mass. Recent studies using GF mice demonstrate that the GM is a regulator of bone mass, and we propose that the inhibitory effect of the GM on bone mass is mainly mediated via effects on the immune system, which in turn regulates osteoclastogenesis. However, we cannot fully exclude that other mechanisms such as altered calcium metabolism and regulation of gut-derived serotonin also contribute. Diet is an environmental factor affecting the composition of the GM. Studies have shown that antibiotic, probiotic, and prebiotic treatments, which have an impact on GM composition, regulate bone metabolism. We propose that the GM might be a promising novel therapeutic target for osteoporosis.
in sex-steroid-deficient mice. Moreover, Leuprolide increased the frequency of TNFa+CD4+ and TNFa+CD8+ T cells in the bone marrow of CONV-R mice, but not of GF mice. These findings suggest that the GM is required to shape the immune system to respond to sex-steroid deficiency. The authors conclude that the GM plays a significant role in inducing bone loss and increasing bone turnover in sex-steroid-deficient mice by providing the antigens required for bone marrow T cell expansion and increased TNFa production [41]. Furthermore, they propose that the GM composition may be involved in the regulation of the magnitude of bone loss experienced by postmenopausal women. One may speculate that an increased inflammatory status caused by an unfavorable GM composition would result in a greater bone loss when women lose the immunosuppressive effects of estrogen after menopause. GM composition and bone loss The GM composition can be modulated by environmental factors, such as diet and treatments with antibiotics, prebiotics, and probiotics (Figure 1). A role of the GM in bone mass regulation is supported by a study demonstrating that subtherapeutic antibiotic treatment to mice at weaning increases bone mass after 3 weeks of exposure [37]. Although the low dose of antibiotics in this study did not cause a significant alteration in bacterial count, it caused shifts in the composition of the GM. In a followup study, low-dose penicillin delivered from birth or from weaning caused an increased BMD in adult female mice [38]. Furthermore, tetracycline treatment has been shown to prevent bone loss after ovx, partly due to inhibited bone resorption [43], and in a similar manner as described for the sex-steroid-deficient GF mice [41]. In another study, tetracycline treatment improved mechanical 3
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Review properties of bone after ovx [44]. These studies demonstrate that antibiotic treatment has the capacity to influence both the GM composition and bone mass, supporting the notion that the GM is a regulator of bone homeostasis. Probiotics and prebiotics Low-dose antibiotic treatment has been used as a growthpromoting agent in farm animals for decades [45]. Because of residuals in meat products and the development of antibiotic-resistant bacteria, this practice is now banned in Europe. This has raised the interest in using other treatments such as probiotics and prebiotics to improve health status and growth. Probiotics are defined as live commensal microorganisms such as bacteria or fungi that when administered in adequate amounts can confer a health benefit on the host such as improved intestinal function and integrity of the intestinal lining. They may also positively affect immune responses in the host gastrointestinal tract to promote health. Several probiotic strains of commensal bacteria exist that are used in experimental settings and some are currently being examined to treat inflammatory bowel disease [46]. Prebiotics are non-digestible food ingredients such as oligosaccharides and dietary fiber that stimulate the growth and/or activity of beneficial bacteria in the digestive system in ways claimed to be beneficial to health [47]. The suggested underlying mechanisms conferring these beneficial effects are manifold and include increased solubility and absorption of minerals as well as anti-inflammatory effects [48]. The effect of probiotics on bone mass has been evaluated in chickens treated during 6 weeks with a diet supplemented with or without two probiotic strains, demonstrating that the probiotic supplementation increased bone mass in the tibia [49]. As several studies indicate that the GM modulates the magnitude of the bone loss in sex-steroid-deficient female mice, it was hypothesized that treatment with probiotics might protect mice from ovx-induced bone loss [41,43,44]. A single (Lactobacillus paracasei DSM13434; L. para) or a mixture (L. paracasei DSM13434, Lactobacillus plantarum DSM 15312, and L. plantarum DSM 15313; L. mix) of probiotic strains was selected based on their anti-inflammatory properties, as shown in an earlier study [50], and was given in the drinking water during a 6-week period to mice, initiated prior to ovx. Both the L. para and the L. mix treatments were shown to protect the mice from ovx-induced bone loss and bone resorption [51]. Subsequent mechanistic studies revealed that the probiotic treatment reduced the expression of two inflammatory cytokines, TNFa and IL-1b, and increased the expression of osteoprotegerin (OPG), a potent inhibitor of osteoclastogenesis, in bone of ovx mice. A bone-protective effect of probiotic treatment using a different strain in ovx mice has also been reported. Britton et al. tested the effect of Lactobacillus reuteri, a commensal bacterium that also secretes beneficial immunomodulatory factors, on ovx-related bone loss. Treatment with L. reuteri altered the GM composition and prevented ovx-induced trabecular bone loss and bone resorption [52]. In addition, this treatment suppressed the ovx-induced increase in bone marrow CD4+ T cells, supporting the notion that the GM modulates the 4
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immune status in bone, and thereby affects OCL-mediated bone resorption. In a separate study, oral administration of L. reuteri treatment was shown to decrease intestinal inflammation and increase trabecular bone mass in gonadal intact male mice [53]. Taken together, these studies suggest that treatment with probiotics has beneficial effects on the immune status in bone, which in turn results in attenuated bone resorption in both ovx female mice and intact male mice. The effect of the prebiotic galactooligosaccharide (GOS) on GM composition and bone mass has been evaluated in growing rats and humans. In rats, GOS supplementation altered the GM composition, with an increase in the relative proportion of bifidobacteria, and increased the bone mass [54]. In a recent study, GOS supplementation was given to adolescent girls during a 3-week period to test the dose–response effect on calcium absorption [55]. GOS treatment improved calcium absorption and increased the relative proportion of bifidobacteria in the GM similarly to what was earlier observed in growing rats. In addition, Abrams et al. showed that prebiotic treatment enhanced bone mineralization during pubertal growth [56]. The prebiotic used in that study was a mixture of short- and long-chain inulin-type fructans given as a daily food supplement for a period of 1 year, and resulted in a 47% greater increase in BMD compared to the control group. The authors proposed that this effect on BMD was mainly due to improved calcium absorption but the GM composition and possible systemic immune-mediated effects were not evaluated. Collectively, the studies using antibiotic treatment, probiotics, or prebiotics indicate that treatments affecting the GM composition also regulate bone metabolism. One possible mechanism, as suggested by the findings from GF mice, is that the GM exerts its effects on bone mass by influencing the host’s immune system. Concluding remarks and future perspectives In summary, the GM modulates host metabolism and immune status. Recent studies using GF mice demonstrate that the GM is a regulator also of bone mass and it is proposed that the inhibitory effect of the GM on bone mass is mediated via effects on immune status, which in turn regulates osteoclastogenesis. A role of the GM in bone metabolism is further supported by studies demonstrating that antibiotic, probiotic, and prebiotic treatments that impact GM composition regulate bone metabolism. The effect of these treatments is present in periods of rapid growth, such as puberty, when bone mass acquisition is large and during periods of bone loss, such as castration. Recent studies show that the bone loss caused by sexsteroid deficiency is diminished in GF mice and can be prevented by treatment with probiotics. Collectively, these studies suggest that the GM may be a novel therapeutic target for osteoporosis (Figure 1). Treatment with probiotics has already been shown to improve bone mass in rodent models of bone loss, but future randomized clinical trials are required to determine the possible effect of probiotics and other novel therapies modulating the GM composition on bone mass and fracture risk in patients with osteoporosis.
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Review The GF mouse is a useful model to study the role of the GM in bone mass regulation. Genetically engineered GF mouse models, in which selected genes are deleted or overexpressed, can help to identify the molecular mechanism by which the GM affects bone mass. Access to cheaper sequencing and improved bioinformatics tools will allow metagenomic sequencing for the analysis of the GM composition in large prospective cohort studies. This can be used to evaluate the predictive value of the GM composition as a biomarker for low bone mass and fracture risk. In addition, metatranscriptomics and metaproteomics will most likely be used to identify the microbial genes and proteins that have an impact on bone mass and fracture risk. We propose a new cross-disciplinary GM–bone research field called ‘osteo-microbiology’, bridging the gaps between bone physiology, gastroenterology, immunology, and microbiology. Future studies are clearly warranted in this research field to determine if the GM composition might be used as a biomarker for fracture risk prediction and to validate the GM as a possible novel therapeutic target for osteoporosis. Acknowledgments This work was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, COMBINE, the Avtal om La¨karutbildning och Forskning/La¨karutbildningsavtalet research grant in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar So¨derberg Foundation, the Novo Nordisk Foundation, the Magnus Bergvall ˚ ke Wiberg Foundation. Foundation, and the A
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