**2. Osteoporosis**

OP is defined as a systemic and metabolic bone disease characterized by a decreased bone mass per unit of volume and a deterioration of the microstructure of the bone tissue, thus increasing the risk of fracture caused by bone fragility. OP is a female-dominated disease more common in postmenopausal women, with a male/female ratio of 1:6 [7,8]. In physiological conditions, there is a balance between osteogenesis, promoted by osteoblasts, and bone resorption by osteoclasts, regulated by a complex molecular mechanism in which estrogens, parathyroid hormone, vitamin D, and inflammatory cytokines are important factors [9–12]. Osteoblasts secrete the nuclear factor receptor activator ligand κB (RANKL), which interacts with the RANK receptor, a member of the tumor necrosis factor family expressed by osteoclasts and their precursors. The RANK/RANKL interaction, which promotes the differentiation and survival of osteoclasts, is controlled by the soluble decoy

receptor Osteoprotegerin (OPG), a natural inhibitor of RANKL. The alteration of these mechanisms, therefore, leads to the prevalence of the RANK /RANKL interaction and to increased bone resorption with consequent OP [13–16]. It has been observed that the loss of estrogens, a condition typical of the postmenopausal state, increases the expression of proinflammatory cytokines, namely interleukin (IL)-1, IL-6, IL-7, IL-17 TNF α, Macrophage colony-stimulating factor (MCSF), and RANKL by osteoblasts, T cells and B cells. Among these cells, T helper lymphocytes (Th)17 are thought to play a particularly critical role in bone loss associated with estrogen deficiency while, regulatory T cells (Tregs) are capable of producing different cytokines, including Transforming Growth Factor (TGF) beta 1, IL-4 and IL-10, inhibiting bone resorption, and reducing the production of effector cytokines [17–22].

#### **3. Microbiota and Osteoporosis**

The microbiota can, through the regulation of mineral absorption of calcium, phosphorous, and magnesium and the production of incretins, serotonin, and gut-derived factors, influence the health of bone. Many studies have already shown that the intestinal microbiome is closely related to bone metabolism, the absorption of bone minerals in physiological conditions, and to the pathogenesis of OP [8,23–25].

In the studies carried out by Collins et al., it was observed that bone mass was higher in germ-free (GF) mice than in conventional mice; GF mice also had a reduced number of osteoclasts per bone surface and a reduced frequency of CD4+ T cells and osteoclast precursors in their bone marrow [23]. These results varied following colonization of the germ-free gu<sup>t</sup> with a conventional microbiota, suggesting the beneficial action of probiotics in the prevention of OP [26].

Uchida et al. found that, comparing the primary osteoblasts isolated from alveolar bones and scalps of the GF mice and the osteoblasts isolated from the specific pathogen-free (SPF) mice, the last expressed substantially more osteocalcin, alkaline phosphatase (ALP), and insulin-like growth factor-I/II (IGF-I/IGF-II), with a decreased ratio of OPG/RANKL. In the end, the bone density of SPF mice was lower than GF mice, indicating that the gu<sup>t</sup> microbiome has a greater regulatory impact on osteoclasts and bone density [27].

Another important study conducted by Jing-Jing Ni et al. about the valuation of the causal relationship between gu<sup>t</sup> microbiota to bone mineral density (BMD) discovered that an increase in the *Clostridiales* class and in the *Lachnospiraceae* family was negatively correlated to BMD, demonstrating the causal relationship between microbiota and bone development [28].

Significant changes in the intestinal microbiota were observed in patients with OP. In fact, while in healthy controls, the composition of the intestinal microbiota was given by the maximum presence of *Bacteroides*, *Faecalibacterium*, and *Prevotella*, in OP patients, it was possible to observe a variation of the bacterial composition with an increase in the proportion of *Firmicutes* and a decrease in the proportion of *Bacteroidetes* than in healthy people [29].

The diet is therefore essential for the absorption of nutrients and for the composition of the microbiota; high consumption of fats is associated with a reduction in the Bacteriodetes/Firmicutes ratio and metabolic imbalances for the host, as found in patients with OP. On the contrary, a low-calorie diet removes harmful substances, leading to beneficial effects for the host [30,31]. An important role concerning the composition of the intestinal microbiota is played, also, by the use of antibiotics. Prolonged therapy can change the normal composition of the bacterial flora, altering its biological metabolism. This leads to impaired intestinal absorption, especially a deficiency of minerals important for bone health, thus contributing to the development of OP [32].

The link between the microbiota and BMD is now established; in particular, bacterial overgrowth has been associated with malabsorption and the consequent alteration in the metabolism of calcium, carbohydrates, vitamins B and K, essential elements for bone metabolism. Furthermore, a high concentration of probiotics *Lactobacillus reuteri* and *Bifidobacterium longum* facilitate the absorption of calcium, magnesium, and phosphorus, increasing BMD. Some species of Lactobacilli, intervening in the degradation of proteins

present in milk, are the main ones responsible for the beneficial effects of milk found in bone health [33–35].

Alterations of the microbiota are able to lead to a dysfunction of the intestinal barrier with an increase in serum lipopolysaccharide (LPS) and consequent increase in intestinal permeability and osteoclasts survival [36,37].

Moreover, the intestinal microbiota is able to influence bone metabolism both directly, through the production of SCFAs, above all butyrate, and through the influence on metabolic hormones such as serotonin, an important factor in the development and maintenance of bone.

SCFAs play a very important role in bone formation and mineralization, acting on the osteoprotegerin pathway, suppressing the RANKL pathway, and influencing the glucagonlike peptide 1, involved in osteoblast-adipocyte differentiation of bone mesenchymal stem cells [38–43] (Figure 2).

**Figure 2.** SCFAs play a very important role in bone formation and mineralization by acting on the osteoprotegerin pathway, reducing osteoclastogenesis by suppressing the RANKL pathway, and reducing the pH of the intestinal tract by subsequently increasing the absorption of minerals.

A mechanism underlying the changes in the gu<sup>t</sup> microbiota in patients with OP has been hypothesized to involve the immune-inflammatory axis as a key bridge linking the intestinal microbiota to bone metabolism [44–48].

In particular, the microbiota can increase TNF-<sup>α</sup>, one of the activators of the RANK-RANKL pathway, which leads to increased bone resorption by altering bone homeostasis in mice [23].

Finally, the microbiota also appears to influence flavonoids and diethylstilbestrol, estrogens of intestinal origin, whose alteration influences bone homeostasis, being the estrogen deficiency directly involved in the risk of postmenopausal OP [49,50].

It is possible to hypothesize the modulation of the microbiota as a therapy limiting the progress of the OP in addition to conventional therapies. One of the strategies that can be used is the administration of probiotics, live microorganisms that restore intestinal permeability, improve the immune barrier function of the intestine, promote the production of IgA, and inhibit the release of proinflammatory cytokines. Several studies were conducted to evaluate the beneficial action of probiotics on the prevention of primary OP, highlighting complex bone protection mechanisms.

Particularly in vitro studies, *Lactobacillus reuteri* was able to inhibit the differentiation of osteoclasts from monocytic macrophages, releasing an anti-osteoclastogenic factor capable

of modulating osteoclastogenesis [51]. Furthermore, a secreted component of *Lactobacillus reuteri* was sufficient to inhibit TNF α-induced suppression on pre-osteoblastic cells [52]. It has also been shown that *Lactobacillus reuteri* secretes histamine, capable of suppressing the production of TNF α by human monocytoid cells [53]. *Lactobacillus helveticus* and *Lactobacillus casei* have a direct effect on bone cells [54]. In particular, the addition of *Lactobacillus helveticus* fermented milk products to primary bone marrow cultures showed an increased calcium accumulation in osteoblast cultures, suggesting its role as an enhancer in osteoblast differentiation [55]. Both *Lactobacillus rhamnosus GG* (LGG) and the commercially available probiotic supplement reduce expression of TNF α, IL-17, and RANKL in cells isolated from the small intestine and bone marrow in mice who underwent ovariectomy [56].

The mechanisms by which probiotics act in human cells are very complex and not fully explored. The direct action of probiotics on osteoclasts must be considered limited in humans, while probably a key role is played in the intestine. Bacteria have been shown to be essential for the synthesis of numerous vitamins and enzymes required for matrix formation and bone growth, including Vitamin D, K, C, and folate. Furthermore, bacteria of the genus *Bifidobacteria* are capable of producing SCFA, which can reduce the pH of the intestinal tract by subsequently increasing the absorption of minerals. Some studies demonstrate that *Lactobacillus reuteri* is able to suppress the gene expression of proinflammatory and pro-osteoclastogenic cytokines, both in the intestine and in the bone marrow. Probiotic bacteria can directly increase calcium transport across the intestinal barrier by reducing intestinal inflammation [25,51,57–61].

The results showed that probiotic preparations prevent increased intestinal permeability caused by the depletion of sex steroids, thus limiting the production of osteoclasts. This serves as a proof of concept that the gu<sup>t</sup> microbiome and probiotic preparations are involved in trabecular bone loss caused by sex steroid deficiency [8].
