**5. Discussion**

The alteration of the microbiota appears to be a common ground for both OP and FA. In particular, an altered intestinal barrier can be considered common damage to both diseases. The intestinal barrier is defined as a functional unit that constantly balances the antigenic charge of the intestinal lumen with a complex immunological and non-immunological organization of the intestinal mucosa. It performs two fundamental functions for the survival of the individual: allow the absorption of nutrients and defend the body from the penetration of harmful macromolecules mediated by the tight junctions of the apical epithelial cells. It has recently been observed that tight junctions are regulated in their

functioning by cytokines produced in the intestine and can be altered by various factors, including alcohol consumption, dietary imbalances, and the action of bacterial toxins [91]. Alteration of the intestinal barrier and the gu<sup>t</sup> microbiota cause the development of an important inflammatory substrate in the intestine, which leads to FA and the loss of estrogen typical of primary OP. A study by Li et al. found that depletion of hormones increases inflammation in the intestine through a greater antigenic load that crosses the intestinal barrier [22]. Estrogens seem to have an ambivalent role in promoting the development of allergic diseases and the degranulation of mast cells in association with exposure to allergens. Andrè et al. also found involvement of the use of oral contraceptives in the etiology of urticaria and chronic angioneurotic edema [91].

The intestinal microbiota is also able to influence the estrogens circulating level through the secretion of β-glucuronidase, an enzyme that activates them. The integrity of the intestinal barrier, normally preserved by the presence of four phyla, Bacteriodetes, Firmicutes, Actinobacteria, and Proteobacteria, is altered by dysbiosis, in which the reduction in cell-cell junctions increases intestinal permeability, resulting in bacterial translocation inducing a systemic inflammatory state at the basis of various pathological processes. Furthermore, it is important to underline that dysbiosis leads to a reduced deconjugation of estrogens with a reduction in their circulation, leading to the activation of CD4 + T cells. CD4 + T cells produce RANKL, OPG, and TNFα, promoting osteoclast activation and bone absorption through the OPG- RANK-RANKL signal transduction pathway.

There is evidence that the inflammatory process is at the basis of both OP and FA. Several proinflammatory and anti-inflammatory mediators are involved in the immunopathogenesis of FA, in which allergens can stimulate Th1, Th2, and Th17 cytokines in a heterogeneous way. Kara et al. [92] have hypothesized the monitoring of cytokines such as TNFα and IL-6 in the follow-up of patients with FA, while Nadelkopoulou et al. [93] have investigated the role of IL-10 in the treatment of FA. TNF-a and IL-1 are also the main cytokines involved in bone metabolism and bone loss related to estrogen deficiency. The role of IL-33 is being debated [94], its activity contributing to the development of various allergological conditions through its action on mast cells, eosinophils, Th2 cells, Tregs, natural killers, basophils, dendritic cells, and activated macrophages, but at the same time appears to have a protective role on bone by inhibiting RANKL-dependent osteoclastogenesis [91–99].

Bone health is highly dependent on diet and nutritional style that determines the type of microbiota in host organisms. The intestinal microbiome, in fact, contributes to the production of proteins and enzymes related to digestion and energy metabolism since it ferments undigested nutrients transforming them into SCFA, leading to a decrease in intestinal permeability and greater absorption of minerals such as calcium. There are several factors linked to the microbiota that unite OP and FA; in particular, a key role is played by SCAF with immunomodulatory and anti-inflammatory effects, exercised through the promotion of immune tolerance, the production of IgA and IgM, the reduction in the production of proinflammatory cytokines including IL-1β, IL-6, and IL-17 [41], and the production of anti-inflammatory IFNγ and IL-10 with consequent expansion of Treg cells and suppression of proinflammatory Th17 and Th2 cells.

The strong influence of the diet on the microbiota and consequently on the OP and allergic manifestations has led to evaluate the fundamental role of nutrition. In fact, a diet rich in fat can reduce the absorption of essential elements for bone health, including vitamin D, K, C, and folate. Moreover, the high-fat diet of Western countries is one of the factors that can contribute to the increased prevalence of allergies in Western countries [100].

Diet-induced obesity has been demonstrated to be a factor of increased susceptibility for FA, and in particular, the microbiota associated with the high-fat diet was found to be able to increase the propensity for FA as evidence of the connection between diet-microbiota and FA [101]. It has been reported that heat-killed lactic acid bacteria (LAB) increased the percentage of peripheral CD4 + CD25 + Foxp3 + Treg cells and relieve symptoms in the pollen season when administered to patients with mild Japanese cedar pollinosis. Although not through the microbiota, LAB is thought to act directly on the immune system. In particular, increased Treg, along with SCFA, is considered a promising target for improving both allergies and bone metabolic balance [102]. Roduit et al. analyzed the levels of SCFA in fecal samples of 301 children at the age of 1, reporting that children with the highest levels of butyrate and propionate were less likely to suffer from asthma by the age of 3 and 6 years and showed significantly lower allergic sensitization with a decrease in food allergy risks and allergic rhinitis diagnosis. More recently, Cait et al. examined the role of bacterial butyrate production in the gu<sup>t</sup> during early childhood in the development of atopic disease and concluded that the lack of genes encoding key enzymes for both the breakdown of carbohydrates and the production of butyrate was the basis of allergic sensitization [1].

Although OP and allergies are two conditions with a high prevalence in the general population and the relationship between fracture risk and allergic diseases such as asthma, atopic dermatitis, urticaria, FA is now well established, to date, we do not ye<sup>t</sup> have adequate epidemiological studies on the prevalence of allergies in patients with OP [103–109]. This probably partially depends on the recent recognition of their interrelationship other than the difficulty in including all together so many "allergic" pathologies with such different peculiarities. The currently available data in the literature refer to single pathologies and limited series. Furthermore, the available studies evaluate the presence of OP in allergic diseases and not the prevalence of allergies in patients with OP. Even in recent studies conducted to evaluate the prevalence of comorbidities in subjects with OP, no data relating to allergies emerge [110], probably because there is still not enough awareness of the influence of allergic disease on bone health. Furthermore, the retrospective analysis conducted in Italy on 64.852 subjects at high risk for fracture collected between 2016 and 2020, through the DeFRAcalc79, does not take into account allergic diseases among the variables for calculating the risk of OP [111].

Vitamin D plays an important role in the pathogenesis of OP and also in the regulation of intestinal tight junctions, leading to the hypothesis that its deficiency may compromise the integrity of the barrier or induce alterations in the composition of the intestinal microbiota, increasing the risk of FA and of OP. Vitamin D, in fact, is important in the maintenance of bone health through the regulation of serum calcium homeostasis. The lack of vitamin D increases bone resorption in order to maintain the right serum calcium levels, making up for the lack of calcium reabsorbed by the gu<sup>t</sup> induced by vitamin D deficiency [112].

Sardecka-Milewska et al. found that children with cow's milk allergy have lower serum concentrations of vitamin D than healthy children [113]. The role of vitamin D in the development of FA is further confirmed by Koplin et al., who documented an attenuated association between low serum levels of vitamin D and food allergy only in subjects with polymorphisms associated with lower levels of vitamin D-binding protein. This involvement of vitamin D in FA could be linked to the ability of vitamin D to induce the expression of IL-10 by Treg cells, leading to oral tolerance and its maintenance [114,115].

Finally, it is also essential to remember the relationships between microbiota and microRNA (miRNA). The latter are small non-coding RNAs capable of regulating gene expression. The importance of the role of miRNAs in many pathological conditions [116–118], including allergies [119] and OP [120,121], is emerging. Being able to fully understand the relationship between miRNA and microbiota could allow to have new disease markers and pave the way for new targeted and personalized therapeutic strategies [122].

The importance of the unaltered microbiota is underlined by the fact that the growing tendency to use antibiotics leads to impaired intestinal absorption with a deficiency of minerals important for bone health, thus contributing to the development of OP, on the other hand, the use of antibiotics compromises the development of oral tolerance mechanisms leading to increased development of FA.

On this basis is founded the use of prebiotics and probiotics for beneficial modulation of the intestinal microbiota both in OP and in allergological pathologies. For example, it has been shown that LGG is able to reduce the expression of TNF α, IL-17, and RANKL in cells isolated from the small intestine and bone marrow of mice, decreasing bone resorption, and also have desensitizing effects in cow's milk and peanut allergy [45,88]. The data in this review based on the current literature highlight how the microbiota and some bacterial species can influence the propensity to develop diseases, including allergies and OP. In particular, the supplementation of beneficial bacteria and diet corrections seems to improve the outcome and prevent the onset of these diseases.
