Abstract
Background:: Knowledge of the complex interplay between gut microbiota and human health is gradually increasing as it has just recently been a field of such great interest. Summary:: Recent studies have reported that communities of microorganisms inhabiting the gut influence the immune system through cellular responses and shape many physiological and pathophysiological aspects of the body, including muscle and bone metabolism (formation and resorption). Specifically, the gut microbiota affects skeletal homeostasis through changes in host metabolism, the immune system, hormone secretion, and the gut-brain axis. The major role on gut-bone axis is due to short-chain fatty acids (SCFAs). They have the ability to influence regulatory T-cell (Tregs) development and activate bone metabolism through the action of Wnt10. SCFA production may be a mechanism by which the microbial community, by increasing the serum level of insulin-like growth factor 1 (IGF-1), leads to the growth and regulation of bone homeostasis. A specific SCFA, butyrate, diffuses into the bone marrow where it expands Tregs. The Tregs induce production of the Wnt ligand Wnt10b by CD8+ T cells, leading to activation of Wnt signaling and stimulation of bone formation. At the hormonal level, the effect of the gut microbiota on bone homeostasis is expressed through the biphasic action of serotonin. Some microbiota, such as spore-forming microbes, regulate the level of serotonin in the gut, serum, and feces. Another group of bacterial species (Lactococcus, Mucispirillum, Lactobacillus, and Bifidobacterium) can increase the level of peripheral/vascular leptin, which in turn manages bone homeostasis through the action of brain serotonin.
Inhibition of T-cell trafficking from the gut to the bone marrow with butyrate directly or indirectly (prebiotic, probiotic supplementation) may represent new strategies to prevent bone loss or stimulate bone anabolism.
Introduction
The gut microbiota (GM) consists of all the living commensal, symbiotic, and pathogenic microorganisms that reside in the gastrointestinal tract [1]. These trillions of microorganisms that line the mucosal surfaces of the gut are transferred from the mother at the time of birth and may undergo several variations being influenced by the mother’s age [2], diet during pregnancy, drug use, or pathological conditions. GM plays a crucial role in host health by establishing a series of mutually beneficial interactions [3, 4] and affects the host on many natural processes of the organism such as intestinal function and physiology, intestinal production and absorption, energy extraction, endocrine system, host growth, metabolic functions, immune potential, and inflammatory processes [5‒9].
Furthermore, it is now known how the gut is in communication with the brain by establishing a gut-brain axis through the connections that the GM establishes between the barriers of our organism [10]. GM disruption has therefore been associated with the pathogenesis of several complex immunological, gastroenterological, rheumatological, cardiological, and neurological diseases [11‒17].
Altered GM composition can also affect muscle and bone metabolism (formation and resorption) [18] (shown in Fig. 1). Osteoporosis is a metabolic bone disease characterized by a decrease in bone mineral density (BMD) with alterations in bone microstructure that leads to an increased incidence of fractures [19]. Bone mass increases after birth to reach a peak in adulthood and is influenced by lifestyle, age, and biological sex [20]. Bone metabolism depends on the balance between bone formation and resorption. When bone formation predominates, it is referred to as anabolism, whereas when the metabolism is directed by bone resorption, it is referred to as catabolism [21, 22]. The hormonal environment, the immune system, the metabolic pathways, and, last but not least, the GM can influence bone metabolism and direct it toward one of these two pathways [23]. Specifically, an alteration of the GM, defined as dysbiosis, can have osteoporotic effects on the bone [24]. Microbial dysbiosis reduces intestinal calcium absorption and dysregulates osteoclast (OC) activity by increasing serum levels of insulin-like growth factor 1 (IGF-1) [25]. Moreover, alterations in the microbiota also reduce bone strength and quality and influence the osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL) pathway in osteoclasts [26‒28]. Based on this, we have reviewed and summarized the mechanisms by which the GM can influence bone metabolism and how the use of prebiotics and probiotics can represent a treatment for bone damage [29‒31]. Indeed, dietary supplements and probiotics could play a role in managing osteoporosis, alongside the already well-known medical and lifestyle interventions.
Gut-Bone Axis Pathways
Dysbiosis and the Immune System
The GM serves as a regulator of BMD through various mechanisms [32]. Several interventions, such as fecal microbiota transplantation in germ-free mice and antibiotic treatment in conventionally raised mice, have demonstrated that alterations in the GM can significantly influence bone metabolism. Similarly, physiological processes like “maternal vertical transmission” and “cohabitation transmission” affect bone remodeling by modifying the GM composition [33‒40].
The GM affects bone metabolism through both direct and indirect mechanisms. Directly, it influences bone health by releasing extracellular vesicles (EVs) and microbial metabolites, including short-chain fatty acids (SCFAs), polyamines, and hydrogen sulfide. Indirectly, the GM affects bone remodeling by interacting with immune cells, such as T helper 17 (Th17) cells and regulatory T cells (Treg), as well as through the hormonal system by influencing the production of hormones like estrogen and parathyroid hormone [41].
Intestinal dysbiosis is closely linked to increased intestinal permeability, which is marked by a decrease in the protein components of intestinal tight junctions. This condition leads to bacterial translocation, chronic inflammation, and the migration of inflammatory cells [42, 43]. This dysfunction is known as “leaky gut” [44, 45]. A permeable gut facilitates the translocation of harmful microorganisms and microbial components into the systemic circulation, which can then reach other organs, including bones, and modulate their functions. For instance, lipopolysaccharides are potent bacterial toxins that, once translocated from the intestine through the bloodstream, can reach the bone, causing resorption. Lipopolysaccharides stimulate the production of pro-inflammatory cytokines: macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF-α), interleukin (IL)-1, and IL-6, maintaining an inflammatory environment [46]. IL-6 enhances OC formation by upregulating RANKL, while both M-CSF and RANKL induce OC differentiation. Chronic inflammation, the cause of chronic inflammatory bowel diseases, is associated with bone destruction. Various chemokines and inflammatory cytokines in the femur, including granulocyte colony-stimulating factor (G-CSF), TNF-α, IL-12p40, MCP-1/CCL-2, RANTES/CCL-5, and keratinocyte-derived chemokines/CXCL1, are significantly elevated in multiple colitis models, leading to the proliferation of OC precursor cells [47]. Th17 cells facilitate OC differentiation even in the absence of external osteoclastogenic factors and can produce other inflammatory chemokines such as M-CSF, IL-17, and RANKL. Additionally, they stimulate bone marrow mesenchymal stem cells to generate OC precursor cells, which results in increased bone resorption [48‒50]. Certain bacteria, including segmented filamentous bacteria, Bifidobacterium adolescentis, and Eggerthella lenta, are known to expand Th17 cell colonies [51].
On the other hand, Treg cells suppress osteoclastogenesis and enhance bone formation by secreting anti-inflammatory cytokines like IL-4, IL-10, and transforming growth factor-β (TGF-β) [52, 53]. IL-10, an inhibitory cytokine, reduces the expression of RANKL and M-CSF while increasing the secretion of OPG, thereby inhibiting OC differentiation and maturation. TGF-β is essential in regulating various stages of osteoblast (OB) differentiation, promoting the proliferation and early differentiation of OB progenitor cells, and enhancing bone matrix production. Additionally, Treg cells can induce CD8+ T cells to secrete the Wnt ligand Wnt10b, which promotes bone formation through Wnt signaling activation in OBs [54]. Some metabolites from GM fermentation, such as butyrate (an SCFA), can stimulate Treg production and support bone formation [55, 56] (shown in Fig. 2).
GM influences bone remodeling through the immune system. Th17 and Treg cells have opposite effects on bone homeostasis. Th17 cells induce OC proliferation, leading to osteoclastogenesis. In contrast, Treg cells induce osteosynthesis by increasing the secretion of inflammatory cytokines such as IL-4, IL-10, and TGF-β that promote OB maturation.
GM influences bone remodeling through the immune system. Th17 and Treg cells have opposite effects on bone homeostasis. Th17 cells induce OC proliferation, leading to osteoclastogenesis. In contrast, Treg cells induce osteosynthesis by increasing the secretion of inflammatory cytokines such as IL-4, IL-10, and TGF-β that promote OB maturation.
The GM also impacts bone homeostasis by regulating other components of innate immunity. Pathogen recognition receptors (PRRs) are the first line of host defense through the recognition of pathogen-associated molecular patterns. Among the PRRs, Toll-like receptors 5 (TLR5), located in epithelial cells, can indirectly modulate bone metabolism. Activation of TLR5 increases the RANKL/OPG ratio in OBs, stimulating OC formation and promoting bone resorption. Nucleotide-binding oligomerization domain (NOD) proteins 1 and 2 are other PRRs located in the cytoplasm. NOD1 and NOD2 bind to peptidoglycans on the surface of Gram-negative and some Gram-positive bacteria (Bacillus, Listeria). When bacteria bind to NOD, they induce inflammation and stimulate NF-κB activity. Activation of this pathway induces a pro-inflammatory response, which activates RANKL, a promoter of bone resorption [57‒60]. Given this intricate and varied relationship between the immune system and bone metabolism, recent studies refer to “osteoimmunology” to define immune cells or immune-related factors that modulate skeletal development [1, 61].
Gut Microbiome-Related Metabolites
SCFAs are among the most significant metabolites produced by the GM and can diffuse from the gut into the systemic circulation, playing a crucial role in regulating bone metabolism [62]. They are signaling molecules that mediate interactions between daily diet, GM, and host health, playing critical roles in immune, metabolic, and endocrine aspects [63]. SCFAs can influence the immune system by altering gene expression, cellular chemotaxis, differentiation, proliferation, and apoptosis. SCFAs, mainly composed of acetic, propionic, and butyric acids, are produced by the fermentation of indigestible dietary fibers by the GM. Specifically, SCFAs modulate bone metabolism by influencing intestinal calcium absorption, increasing villous structure and surface area of the small intestine epithelium, and enhancing the expression of calcium-binding proteins [64]. Additionally, SCFAs can lower the pH in the intestinal cavity, improving mineral solubility and making calcium more readily absorbable. Furthermore, they participate in indirect regulation by modulating IGF-1, leading to higher bone formation rates and trabecular bone mass in mice [65]. Finally, SCFAs can stimulate Treg generation, ensuring immune homeostasis by inhibiting intestinal inflammation and OC differentiation.
SCFAs are among the most significant metabolites produced by the GM and can diffuse from the gut into the systemic circulation, playing a crucial role in regulating bone metabolism
Bile acids also play a fundamental role in lipid and bone metabolism. They are divided into primary bile acids, forming bile salts in the liver, and secondary bile acids. The GM can alter the quantity and type of primary bile acids, resulting in metabolic effects on bones. For instance, as signaling molecules, bile acids not only contribute to the absorption, transport, and distribution of fats and fat-soluble vitamins, but also are closely associated with energy metabolism modulation, thereby suppressing excessive proliferation of the GM [66]. Secondary bile acids play a crucial role in modulating the inflammatory and immune response induced by TNF-α. 3-oxoLCA inhibits Th17 cell differentiation by directly binding to the key transcription factor retinoid-related orphan receptor gamma t (RORγt), while isoalloLCA promotes Treg differentiation by inducing mitochondrial ROS production, thus participating in bone metabolism modulation [67, 68].
Choline metabolites, specifically trimethylamine N-oxide and indole derivatives, can help prevent BMD reduction by stimulating the production of antimicrobial peptides, mucin proteins, and promoting the proliferation of intestinal villi cells. They help maintain the integrity of the intestinal mucosal barrier by enriching microbial composition and negatively modulating cytokine cascades of inflammatory processes [69, 70].
EVs and Bone Remodeling
Most organisms on Earth, including eukaryotes, Gram-negative and Gram-positive bacteria, and archaea, possess tiny vesicles in the extracellular environment [71, 72]. These vesicles are spherical with a proteolipid bilayer and contain specific subsets of bioactive proteins, lipids, nucleic acids, and metabolites [73, 74]. Gram-negative and Gram-positive bacterial EVs perform various physiological and pathological functions in bacteria-bacteria and bacteria-host interactions, either through direct activation of target cells by the vesicles or by transferring functional vesicular cargo to recipient cells [75‒77]. The host-bacteria interactions mediated by Gram-negative bacterial EVs can trigger a range of reactions – non-immunogenic, pro-inflammatory, or cytotoxic – depending on the type of cell affected, the bacterial species, and the quantity of vesicles, evidently also playing a role in bone metabolism.
Endocrine System Related to Intestinal Microbiome and Bone Remodeling
Most importantly, serotonin (5-hydroxytryptamine [5-HT]), a hormone and neurotransmitter, plays a crucial role among indole derivatives by influencing the dynamic balance between bone formation and resorption [78]. It has been discovered that brain-derived 5-HT promotes bone formation by inhibiting peripheral autonomic nerves, while gut-derived 5-HT inhibits bone formation by reducing OB proliferation [79].
Additionally, polyamines, such as humic acid, spermidine, and spermine, can modulate gene expression, promote epithelial cell proliferation, maintain intestinal mucosal barrier function, and modulate bone metabolism by influencing immune system function [80, 81]. Recent studies have shown that the hormone leptin could also regulate bone homeostasis by modulating brain serotonin levels. Leptin is an adipocyte-derived hormone initially recognized for its role in energy expenditure and homeostasis [82]. Leptin binds to its receptor expressed in the brain, leading to the inhibition of serotonin receptors and a decrease in brain serotonin levels. The beneficial effect of brain serotonin on bone mass is thus inhibited by leptin [83]. Therefore, inhibiting intestinal 5-HT biosynthesis could become an effective approach in preventing and treating osteoporosis and bone demineralization.
Role of Dietary Supplements and Probiotics in Modulating Microbiota and Bone Health
A diet rich in fiber, fermented dairy products, and polyphenols is the simplest and primary means to cultivate a diverse and substantial microbial composition in the gut, influencing bone health and well-being [49, 84]. The Mediterranean diet, for instance, in contrast to a high-fat diet, benefits human GM by reducing the Firmicutes-to-Bacteroides ratio and increasing SCFA levels [85]. Traditional therapeutic strategies for preventing BMD loss, such as anabolic and anti-resorptive agents [86], have significant long-term side effects. Therefore, given the crucial role of GM in bone modulation, efficient alternative solutions targeting GM are being developed. These include the use of prebiotics, probiotics, synbiotics, and fermented foods.
A diet rich in fiber, fermented dairy products, and polyphenols is the simplest and primary means to cultivate a diverse and substantial microbial composition in the gut, influencing bone health and well-being
Probiotics are “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [87]. They are found in yogurt, dairy-based foods, powders, capsules, or oral solutions. These include Lactobacillus, Bifidobacterium, Escherichia, Enterococcus, Bacillus subtilis, and yeasts like Saccharomyces. Probiotics can directly affect immune and epithelial cells by producing genes and metabolites that promote bone formation and health, improve the intestinal mucosal barrier function, reduce inflammatory cytokine cascades, lower intestinal pH, stimulate calcium absorption, and resist harmful external agents [88, 89]. Specifically, probiotics reduce the expression of inflammatory cytokines (TNF-α and IL-1β) and enhance the expression of OPG in bone [90]. A clinical study on Lactobacillus reuteri showed that this probiotic increases serum 25-OH-VD levels in healthy subjects and enhances calcium function and absorption in the body [91]. Supplementation with Lactobacillus rhamnosus GG increases trabecular bone volume. The butyrate produced in the gut by Lactobacillus rhamnosus GG induces the expansion of intestinal Tregs, leading to an increase in Wnt10b, an anabolic bone ligand [92, 93].
Prebiotics are dietary components fermented by GM, resistant to gastric acid and hydrolytic enzymes, and nondigestible and nonabsorbable by the intestine. They are mostly products of enzymatic conversion of sugars, such as galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, resistant starch, xylooligosaccharides, and lactulose. Prebiotics can also effectively regulate the composition and activity of the host’s GM [94]. Specifically, they increase the concentration of bifidobacteria and lactobacilli in the gut, which are associated with proliferation and increased activation of osteoblasts [95‒97]. Administration of FOS or GOS can optimize biodiversity and increase SCFA concentrations in GM. Prebiotics share the ability to be converted into SCFAs like acetate, propionate, and butyrate by GM, increasing their serum levels and lowering pH. This leads to greater absorption of minerals like magnesium and calcium in an acidic environment [98, 99]. Butyrate also acts as a growth factor for enterocytes and colonocytes. Moreover, SCFAs generated by prebiotics stimulate Treg cells in the colon, limiting inflammation [100] and modulating the synthesis of IGF-1, which is involved in bone remodeling [101].
Diet influences GM and intestinal function [18]. A fiber-rich diet with GOS and FOS supplementation and the use of probiotics can increase Bifidobacterium species, optimize GM composition and abundance, increase SCFA content, lower intestinal pH, and promote calcium absorption and bone retention [102].
Nutritional Interventions and Therapeutic Strategies to Preserve Bone Mass in Children
A study in severe acute malnutrition in Bangladeshi children showed that the infant treated with therapeutic foods showed reduced plasma levels of an osteoclastic activity biomarker without affecting osteoblastic activity biomarkers. Germ-free young mice were administered cultured bacterial strains from a rickety 6-month-old infant and fed a diet mimicking the donors’ population. Purified sialylated bovine milk oligosaccharides, structurally similar to human milk, were added. The results were increased femoral trabecular bone volume and cortical thickness, reduced osteoclasts, their bone marrow progenitors, and osteoclastogenesis regulators and mediators [103].
Another randomized, double-blind, controlled clinical trial was conducted in the Philippines. Children aged 2–3 years received for 6 months either an experimental young child formula with synbiotic (n = 91) containing L. reuteri DSM 17938 (at 108 CFU/day) + GOS (4 g/L) or minimally fortified powdered milk mimicking local traditional milks (control milk, n = 91). Children were advised to consume two servings daily. Tibial quality was assessed at baseline, 3, and 6 months by measuring ultrasound speed. A mixed linear model with repeated measures and fixed effects for treatment group, sex, visit, visit × treatment interaction, and baseline values for ultrasound speed, blood vitamin D levels, and BMI was applied. Bone turnover markers (CTX and P1NP) and microbiome composition and function were analyzed using ELISA and shotgun metagenomics, respectively. Results indicated that consuming the synbiotic L. reuteri + GOS formula improved bone strength and quality by enhancing bone turnover and inducing microbiome changes supporting bone quality improvement in healthy children.
Recent studies have shown that GM transplantation from young rats reduced bone loss in elderly rats with senile osteoporosis, decreasing bone turnover markers. Additionally, transplanted GM improved intestinal microbiome composition and barrier function [104].
Conclusion
It is certain that GM regulates skeletal homeostasis through changes in host metabolism, immune system, and hormonal secretion. Therapeutic intervention with dietary supplements like prebiotics, probiotics, postbiotics, and synbiotics should be further explored as useful adjunct treatments to modulate bone metabolism, the gut-bone axis, and support bone health.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This article is an editorial for the 101st NNI/WNSC workshop, for which FI received an honorarium.
Author Contributions
F.I. and A.S. conducted literature research and synthetized information. A.S. and F.I. wrote the manuscript. F.I. revised the whole manuscript. All the authors read and approved the final version of the manuscript for publication.