Introduction

The commensal gut microbiota (GM) in our intestine performs various useful functions by modulating both the metabolism and immune status of the host [1]. The importance of the GM for both health and disease has been an active area of research in the recent past. The composition of GM is modulated by a number of environmental factors such as diet, antibiotic treatments or pathogens. Prebiotics on the other end are non-digestible food supplements that exert a health-promoting effect on the host by selectively stimulating the growth and activity of various bacteria residing within the gastrointestinal tract [2]. Probiotics literally means “for life” and the definition of probiotics has been varied since it was first coined [3]. Probiotics are mostly commensal bacteria residing in the gut. Probiotics are defined as live commensal microorganisms such as bacteria or fungi which when administered in adequate amounts can confer a health benefit on the host by altering the composition of the GM. Probiotics also positively affect the immune responses in the host gastrointestinal tract thereby promoting health. Some of the probiotics are bacterial components comprising of normal human intestinal flora which produce various beneficial end products such as short chain fatty acids and lactate metabolism. These bacterial populations have the potential to treat different clinical conditions [4], including osteoporosis, diabetes, fatty liver disease, inflammatory bowel disease (IBD) and cardiovascular disease (CVD) [5,6].

One of the key bone diseases associated with aging and postmenopausal condition is osteoporosis which affects more than 75 million people in Europe, Japan, and the USA. It is estimated that by the end of 2020 more than 60 million men and women will be suffering from osteoporosis [7]. Menopause is a major risk factor for osteoporosis. In fact fifty percent of the women population over the age of 50 will experience an osteoporosis-related fracture in their lifetime [8]. Also there is elevated risk of wrist, hip, or spine fracture due to osteoporosis which is estimated to be parallel to the risk of heart disease [9].The current review specifically focus on the vital role of probiotics in regulating bone health, which itself is modulated by the host immune system.

Gut microbiota and immune system

A cross talk between the host mucosal innate immunity and GM augments the growth, survival and homeostasis of the gut ecosystem. The GM plays a primary role in the induction, education, and function of the host immune system. On the other hand the immune system has principally evolved as a means to maintain the symbiotic relationship of the host with these highly diverse and evolving microbes. The resulting microbiota-immune system association allows the induction and maintenance of various regulatory pathways involved in tolerance to these diverse antigens [10]. A characteristic feature of the innate immunity is its ability to distinguish between beneficial and pathogenic microbes by the help of various “pattern recognition receptors” (PRRs). The toll-like receptors (TLRs) enable the cells to recognize highly conserved and diverse molecules on the surface of microorganisms, collectively called as pathogen-associated molecular patterns (PAMPs) [11]. Since these molecules e.g. lipopolysaccharides, peptidoglycans, flagellin and others, are also present on various commensal bacteria, they are often called as microbe-associated molecular patterns (MAMPs). The GM regulates the intestinal immunity by modulating the expression of TLRs on immune cells surface through MAMPs. The GM shapes the T cell repertoire in the lamina propria along with modulating the homeo­stasis of the host [12]. These mucosal T cells function as important mediators for not only the defense against intestinal pathogens, but they also help in wound healing, barrier repair and regeneration at sites of infection and injury [13]. T cells depending upon the environment milieu, which is regulated by the presence of various cytokines can either drive a pro-inflammatory (TH1, TH2 and TH17 cells) or an anti-inflammatory immune response (regulatory T cells-Tregs) [14]. Antibodies (mainly IgA) specific for intestinal bacteria is secreted with the help of intestinal dendritic cells (DCs) that sample the various bacteria penetrating the gut epithelium. These bacteria-laden DCs interact with B and T cells in the Peyer's patches and activate B cells to produce IgA specific against the intestinal bacteria [15]. Thus, IgA (a key player in barrier homeostasis) secreted by plasma B cells residing in the lamina propria, binds to the microbial antigens present in the gut lumen and prevent there translocation and infection [16]. The dif­ferentiation of B cells into IgA-producing plasma cells is induced via the stimulation of TLR5 on DCs by GM derived flagellin [17]. The intestinal microbiota is also responsible for modulating the development of invariant NK T cells (iNKT cells) responsible for various inflammatory immune responses [18]. Thus both the intestinal innate and adaptive immunity go hand in hand in maintaining homeostasis of the gut microenvironment [13].

Interplay between bone and immune system

The skeleton is continually being remodeled by the activity of bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCLs) [20]. OBs are derived from mesenchymal stromal cells whereas OCLs are derived from hematopoietic stem cells which are also the progenitors of immune cells [21]. OCLs are distinctively derived from myeloid-monocyte lineage of hematopoietic cells under the influence of local microenvironment. Macrophage colony stimulating factor (M-CSF) induces proliferation and up-regulated expression of receptor activator of nuclear factor-κB (RANK) on OCL precursor cells. This results in binding of RANK ligand (RANKL) on its receptor RANK present on the surface of OCL precursors leading to enhanced osteoclastogenesis [21]. Any imbalance between the processes of osteoclastogenesis and osteoblastogenesis results into the induction of various inflammatory bone diseases such as rheumatoid arthritis (RA) and osteoporosis [22]. The effect of inflammation on bone loss is well established. In various auto-immune diseases such as rheumatoid arthritis, osteoclastic bone resorption is driven mainly by the secretion of various inflammatory cytokines produced by T cells [23]. During menopause the resulting estrogen deficiency leads to enhanced formation and survival of OCLs. This effect is in part modulated due to loss of the immunosuppressive effects of estrogen, resulting in enhanced production of cytokines by T cells (Th17 cells) thereby promoting osteoclastogenesis [24].

Probiotics and bone health

Recent studies demonstrate that the GM regulates bone mass and this effect of the GM on bone mass is mediated mainly via its effect on the host immune system, which in turn regulates osteoclastogenesis. In one such study different strains of bacteria such as Lactobacillus and Bifidobacterium were shown to have the potential to escalate the bone mineral density in ovariectomized (ovx) rats and mice which simulate postmenopausal conditions of osteoporosis [25,26]. In another study the administration of Lactobacillus paracasei (NTU 101) and Lactobacillus plantarum (NTU 102) fermented milk to ovx mice resulted in higher trabecular number compared to ovx and sham-ovariectomized control groups [27]. Administration of Lactobacillus helveticus in male osteoporotic rats is reported to enhance both the bone mineral density and content (BMD and BMC) [28]. Similarly different studies with strains of Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus gasseri described higher bone weight among the probiotic fed group with respect to control groups [29]. A recent study showed a significant reduction in bone resorption by L. reuteri via decreasing levels of tumor necrosis factor (TNF), thereby increasing BMD, BMC, trabecular number and thickness, along-with decreased trabecular space in both vertebral and femoral bones [25]. Strains of Bifidobacterium longum have also been studied to significantly affect bone health [29]. A reduced number of TRAP positive osteoclasts were found in groups fed with fermented broccoli compared to the control groups, while no significant differences were observed in body weight of rats between the groups [30]. Probiotics could also have a potential effect on bone build-up, which can be independent to that of prebiotics. This could occur via microbial synthesis of various metabolites and enzymes or synthesis of several vitamins [31] such as vitamin D, C, or K [32] or folate [33]. Based on these reports one may expect that the optimum combination or use of probiotics and prebiotics would deliver best results, which ultimately depends on the nature of disease or risk being considered. A comparative account of the various studies dealing with the effect of probiotics on bone health is charted in Table1 for ready reference.

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   Table 1:

   Animal studies showing effect of probiotics on bone.
   
   

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   Table 1: Animal studies showing effect of probiotics on bone.
   Probiotic strains	Duration	Possible effect on bone	Subject	Methodology	Author and year
   Bifidobacterium longum (ATCC 15707)	12 weeks	Number of TRAP-positive osteoclasts decreases	Male Wistar rats	Histological methodology	Tomofuji et al. [26]
   Bifidobacterium longum(ATCC 15707)	28 days	(i) Bone weight, thickness Strength of fracture (ii) Increase in Ca, Mg bone content	Male Wistar rats	(i)Stainless steel clipper (ii)Three point Texture Analyser (iii)Plasma emission spectrophotometer	Rodrigues et al. [54]
   Lactobacillus reuteri 6475	4 weeks	(i) ↑increase in BMC (ii) ↑in BMD (iii) ↑in BVF (iv) ↑Trabecular thickness and thickness of distal femur	Healthy male mice	Micro-CT	McCabe et al. [25]
   Bacillus licheniformis and Bacillus subtilis	6 weeks	(i) ↑Medial and lateral wall thickness of tibiotarsi (ii) ↓Medullary canal diameter	Broiler chicks	(i) Dual caliper (ii) Subtracting the thickness of medial and lateral walls from diameter at the diaphysis	Mutus et al. [55]
   Lactobacillus paracasei (NTU101) and Lactobacillus plantarum (NTU102)	8 weeks	(i) ↑Tb. N (ii) ↓Tb. Sp (iii) Femur BMD/NS	Ovariectomized mice	(i) CT system (ii) Skyscan	Chiang and Pan [27]
   Active Lactobacillus casei 393	6 Weeks	(i) ↑ Ca and P in dry femur (ii) ↑BMD and BMC (iii) ↑Bone strength (iv) ↑ Dry weight of femur	Ovariectomized Sprague-Dwaley rats	(i) Vernier caliper (ii) Inductively coupled plasma-optical emission (iii) DEXA (iv) Three-point Texture Analyser	Kim et al. [56]
   Lactobacillus helvecticus LBK-16H	14 Weeks	(i) ↑BMD and BMC (ii) ↑ Femur weight	Male rats induced- osteoporosis aging	DEXA	Narva et al. [57]
   Lactobacillus brevis SBS8803	4 Weeks	(i) ↑BMD and BMC (ii) ↑Bone strength	Ovariectomized Balb/c mice	Micro-CT	Segawa et al. [19]
   NS: not significant, CT: computed tomography, TRAP: tartrate resistant acid phosphatase, Ca: calcium. Mg: magnesium, BMD: bone mass density, Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.sp: trabecular separation, BMC: bone mineral content, DEXA: dual-energy X-ray absorptiometry.

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The anti-inflammatory effects exerted by various probiotic bacteria are thought to be mediated via the induction of Treg cells [34]. Probiotic treatment has been found to reduce the expression of several osteolytic cytokines (TNFα and IL-1β), responsible for altered RANKL/ osteoprotegerin (OPG) ratio in cortical bone. These findings clearly signify that probiotic treatment alters the status of immune cells in the bone microenvironment resulting in attenuated bone resorption observed in ovx mice [35]. TNFα promotes osteoclastogenesis by stimulating expression of RANKL from bone marrow stromal cells and osteoblasts resulting in enhanced osteoclastogenesis from OCL precursor cells via RANKL [36]. OPG inhibits OCL differentiation in a dose dependant manner [37]. These findings clearly delineate the role of probiotic treatment in suppressing osteoclastogenesis. Administration of L. reuteri in mice alters the GM composition and prevents ovx-induced trabecular bone loss and resorption [8]. Also treatment of L. reuteri suppressed the percentage of CD4+ T cells in bone marrow, supporting the notion that GM has immunomodulatory properties and 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 [25]. This decrease in the number of CD4+ T cells in bone marrow in GF mice was a consequence of the impact of the GM on the adaptive immune system, leading to fewer CD4+ cells recirculating in the blood and secondary lymphoid tissue.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation of synovial joints with an increased expression of inflammatory cytokines [38]. TNFα is one of the critical cytokines in the pathogenesis of RA [39]. Patients with psoriatic arthritis and mice with TNFα-induced arthritis have increased numbers of circulating OCL precursors [40]. Treg cells are critical for maintaining self-tolerance and negatively regulate immune responses. Several strains of Lactobacillus have been reported to have therapeutic effect in experimental mouse models of inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis, and are associated with enrichment of Treg cells in the inflamed regions [41]. This inhibitory effect of probiotic L. strains was recently shown to depend on suppressive motifs in the DNA enriched in these strains that potently prevented dendritic cell activation and maintained Treg cell conversion during inflammation [42]. TGFβ is crucial for the activation, differentiation and activity of Treg cells [43]. Interestingly, in one study it was reported that ovx decreases the number of Treg cells in bone marrow in control but not probiotic treated mice. Furthermore, the expression of TGFβ1 was increased by probiotic treatment compared to control groups after ovx, suggesting the role of probiotics in Treg cell differentiation via induction of TGFβ1. In vitro studies have also shown that Treg cells directly inhibit OCL differentiation and function and this effect of Treg cells is stimulated by estrogen [44]. In addition, adoptive transfer of Treg cells decreases the number of OCLs and limits bone loss in ovx mice [45]. Collectively these findings suggest the immune-suppressive effect of probiotic treatment under various inflammatory conditions.

There could be a number of various possible mechanisms which could simultaneously exist for the effect of probiotics on bone. One of the potential effects of probiotics on bone could possibly occurs via the synthesis of vitamins [46].Vitamins like C, D, K, along with folate are involved in metabolism of calcium, a necessary component for bone formation [47]. Moreover, various strains of bacteria produce several short chain fatty acids which decrease parathyroid hormone thereby resulting in an increase in mineral absorption [48]. A recent study showed another mechanism in which intake of probiotics reduce intestinal inflammation and increased BMD, implying that GM have a significant effect on bone health [49]. The bacterial strain Lactobacillus reuteri 6475 have been reported to reduce pro inflammatory cytokine levels systemically, leading to increased bone volume fraction by reducing the expression of pro inflammatory cytokines in both Jejunum and ileum [25]. 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 [50]. Different studies till date point that probiotic treatment reduces the expression of various inflammatory cytokines such as TNFα, IL-1β etc. and subsequently increased the expression of OPG, a potent inhibitor of osteoclastogenesis, in bone of ovx mice resulting in enhanced bone turnover. Currently very few and limited number of available probiotic strains have been explored for their effect on bone physiology, thus a detailed study is still awaited to uncap the huge potential inherent in this field of biology.

Future perspectives

In summary, it appears that the GM modulates both the host metabolism and immune status. Studies using germ free mice (GF) demonstrate that the GM is a regulator of bone mass and proposed that the inhibitory effect of the GM on bone mass is mediated via its effects on immune status, which also regulates osteoclastogenesis. A role of the GM in bone metabolism is further supported by various studies demonstrating that antibiotic, probiotic, and prebiotic treatments that impact GM composition regulate bone metabolism. Probiotics act by altering the composition or the metabolic activity of the GM [39]. The suggested underlying mechanisms for how probiotics contribute to health are manifold including increased solubility and absorption of minerals, enhanced barrier function and modulation of the immune system [51]. Probiotics are increasingly being used worldwide to treat various health issues. Studies indicate that probiotics may be effective in the treatment of some gastrointestinal diseases such as colic, irritable bowel, and inflammatory bowel disease [52]. In addition, probiotics have also showed promise in ameliorating other ailments that are distal to the gut, including eczema, asthma, and allergies [53].

Current studies have shown that bone loss caused by sex-steroid deficiency is diminished in GF mice and can be prevented by treatment with various probiotics. Collectively, these studies suggest that GM may be a novel therapeutic target in osteoporosis (Figure 1). Treatment with probiotics has already been shown to improve bone mass in rodent models of bone loss. Thus future randomized clinical trials are required to determine the possible effect of probiotics along with various other novel therapies which have the potential to modulate the GM composition. Thus the role of GM in regulating the host immune system which in turn regulate bone mass validates the use of probiotics as possible novel therapeutics in treatment of various inflammatory bone conditions such as osteoporosis and RA.

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   Figure 1:

   GM regulates bone remodelling. Based on various recent studies we hypothesize/propose that the inhibitory effect of GM on bone mass is via its regulation of the host immune system (DCs, T, B & iNK cells) and administration of probiotics can escalate this effect. GM is also modulated by different environmental factors such as prebiotics, antibiotics, diet and pathogens.
   
   
   

Acknowledgement

This work was financially supported by a project from UGC Faculty Research Promotion Scheme (FRPS), Govt. of India, sanctioned to RKS. GBT thanks DST, Govt. of India for financial support. RKS acknowledges, Department of Zoology, Dr. H. S. Gour Central University for providing infrastructural facilities and financial support.