**2. Reciprocal interplay between bone and immune cells**

### **2.1 An overview of the "osteoimmunology" field**

The relationship between bone and immune systems has been suggested by pioneering studies reported in the early 1970s and showed that molecules secreted from immune cells were capable to induce OC activation and differentiation [65, 66]. Moreover, early studies in the immunology field, using genetically deficient mice in various immunomodulatory molecules, showed unexpected phenotypes in the skeletal systems under physiological conditions [40, 63, 67, 68]. Actually, we know that bone and immune systems share a variety of molecules, including cytokines, chemokines, transcription factors, and signaling molecules [67]. By interacting with each other in the BM, the bone and immune cells cooperatively conduct a series of bone and immune system functions [67]. Studies conducted on bone and immune

*Perspective Chapter: Breast-Tumor-Derived Bone Pre-Metastatic Disease – Interplay... DOI: http://dx.doi.org/10.5772/intechopen.107278*

phenotypes are revealing the physiological significance of the mechanisms shared by both systems [67], and the interdisciplinary field "osteoimmunology" was created to explore these mechanistic interactions, under physiological or pathological conditions [69].

The RANK/RANKL/OPG molecular system is considered the most important pathway explicitly linking immune and bone tissues [35, 38, 43, 70, 71]. Indeed, several studies are showing that RANK and RANKL, besides being the master regulatory via inducing osteoclastogenesis, also play multiple roles in the immune system, including: (i) differentiation of medullary thymic epithelial cells (mTECs) [72–75]—that act as mediators of the central tolerance process, which self-reactive T cells are eliminated while regulatory T cells are generated; (ii) secondary lymphoid tissue organogenesis—the organization of the microarchitecture of lymph nodes (LNs) [42], formation of germinal centers in gut isolated lymphoid follicles [42] and Peyer's patches [42]; and (iii) fine-tuner of adaptive immune response enhancement of DCs longevity and survival [76], maintenance of immunological memory [77] and B cells ontogenesis [78, 79]. Of note, these molecules are expressed by cells from both systems [63]. OPG, for example, is expressed by mature B cells (accounting alone for almost 40% of OPG produced in BM. Their essential role for bone homeostasis was shown *in vivo*, since B-cell-deficient mice have low bone mass density associated and a marked deficit in BM OPG [80]. This homeostatic balance is achieved by B and T cells interaction, via CD40-CD40L molecules, since mice depleted of CD40 or CD40L co-stimulatory molecules presented a decline in OPG production by B cells and an increase in bone resorption and low bone mass density [80]. Also, mice depleted from T cells showed a complete suppression of OPG production by B cells followed by an increase in osteoclastogenesis and bone loss [80]. Moreover, Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4)—a molecule expressed by T cells that helps keep immune responses in check—binds to CD80/CD86 co-stimulatory molecules expressed by OCs, leading to inhibition of osteoclastogenesis mediated by RANKL or TNF-α [81]. CTLA-4 binding to CD80/CD86 in OCs' precursor cells induces the expression of indoleamine 2,3 dioxygenase (IDO), which in turn degrades tryptophan and leads to OCs apoptosis [82]. Consequently, mice deficient in CD80, CD86, or IDO have increased osteoclastogenesis rates and osteopenic phenotypes [82, 83] demonstrating that CTLA-4 plays important roles in the physiological regulation of bone mass preservation [81–83].

We should be aware that most of these findings were conducted in animal models; however, new indications are emerging to support the reciprocal roles of both systems in human diseases aspects [47, 67, 84]. Despite the more recent observations about the impact of immune cells for bone tissue homeostatic integrity, and vice versa, the interplay between both systems is first spotlighted by studies on bone disorders, triggered by abnormal immune responses activation, like the ones seen in rheumatoid arthritis (RA), post-menopausal osteoporosis, chronic periodontitis, multiple myeloma, fractures, HIV chronic infection, and bone metastases [67].

### **2.2 Role of T and B cells in bone disorders**

T and B cells are derived from the same lymphoid progenitor cell during hematopoiesis and are the main cellular representatives of the adaptive immune system, so called because they do not mount an immediate response to an antigen (Ag).

The Ags are recognized by specific receptors—T cell receptor (TCR) and B cell receptor (BCR), which are diverse at the population level and clonal and unique at individual cellular level. TCRs and BCRs are not conserved and are generated by gene rearrangements during T and B cell ontogenesis. T cells ontogenesis takes place in the thymus, while B cells ontogenesis is in BM—both are primary lymphoid organs.

After maturation inside BM or thymus, B and T cells gain the peripheral blood circulation and enter the secondary lymphoid organs. In lymphoid organs, as LNs and spleen, activated/educated by dendritic cells (DCs), the professional Ag-presenting cell (APC) are found. Through their ability to sense changes in their local environment and respond appropriately, DCs activate T cells by the expression of the Major Histocompatibility Molecules (MHC), in complex with linear, short, peptides Ags (9–20 amino acids long). This complex is recognized by T cells via TCR and CD3 ε and δ, ζ chains accessory molecules and their categorized cluster of differentiation (CD) surface expressed molecules, CD4 or CD8. In addition, T cells concomitantly recognize co-stimulatory molecules and cytokines, which will define their functional differentiation fates, in terms of their expression of master transcription factors and functional cytokines [85]. CD4<sup>+</sup> helper T cells are divided into specialized subsets, known as: (i) T helper 1 (Th1), expressing T bet transcription factor and IFN-γ; (ii) T helper 2 (Th2), expressing GATA-3 transcription factor and IL-4, IL-5, and IL-13; (iii) T helper 17 (Th17), expressing ROR γT transcription factor and IL-17A, IL-17F, IL-22, and IL-26; (iv) T helper 22 (Th22), expressing Runx1 and RORγt transcription factors and IL-22; T follicular (Tfh), expressing B cell lymphoma 6 (Bcl6) transcription factor and IL-21; and (v) T regulatory (Treg) cells, expressing FoxP3 transcription factor and TGF-β and IL-10; while CD8<sup>+</sup> T cells fall into subpopulations, known as: (i) Cytotoxic Type 1 CD8<sup>+</sup> T cells (Tc1), expressing T bet and BLIMP-1 transcription factors, IFN-γ, granzyme, and perforin; (ii) Type 2 CD8<sup>+</sup> T cells (Tc2), expressing GATA-3 transcription factor and IL-4, IL-5 and IL-13; (iii) Type 17 CD8<sup>+</sup> T cells (Tc17), expressing ROR γT and ROR α transcription factors and IL-17A, IL-17F and IL-22 and T reg CD8<sup>+</sup> T cells, expressing IL-10 [85].

B cells are also activated in secondary lymphoid organs, but, in contrast to T cells, they do not need APCs to present their cognate Ags, which will be freely recognized in linear or structural forms. At the beginning of immune responses, B cells secrete immunoglobulins M (IgM) independently of T helper cells. The T-cell-independent response is short-lived and does not result in the production of memory B cells, which will not result in a secondary response to subsequent exposures to the same Ags. However, to induce stronger B cell responses and to generate immunological memory, B cells need help from T follicular CD4<sup>+</sup> T cells (Tfh). Indeed, to enable homing to B cell follicles, Tfh expresses abundant C-X-chemokine receptor type 5 (CXCR5). Another characteristic of Tfh is the expression of CD40 ligand (CD40L), inducible T cell costimulator (ICOS), programmed death-1 (PD-1), and B and T lymphocyte attenuator (BTLA). Tfh cells colocalize with Ag-specific B cells within germinal centers (GCs), which are transient structures located within B cell follicles, in secondary lymphoid tissues, in which somatic hypermutation of immunoglobulin (Ig) variable region genes and selection of high-affinity B cell clones occur. Immunoglobulin class switch (IgA, IgE, and IgG) will be defined by cytokines produced by these different specialized Tfh, at the moment of B cells activation.

### *Perspective Chapter: Breast-Tumor-Derived Bone Pre-Metastatic Disease – Interplay... DOI: http://dx.doi.org/10.5772/intechopen.107278*

It is clear now that the identity of T cell subsets is critical in guiding their role on bone remodeling system, during homeostasis or in pathological conditions [67]. In particular, Th1, Th2, Th17, Th22, and T reg CD4<sup>+</sup> and CD8<sup>+</sup> cells have been shown to influence bone metabolism [67, 86–89]. In the RA scenario—the best studied human disease in osteoimmunology—the importance of Th17 CD4<sup>+</sup> T cells is evident, beginning by their infiltration into the synovium and the association of disease susceptibility with specific variants of T-cells-related genes, such as HLA-DR (MHC class II cell surface receptor encoded by the human leukocyte Ag gene complex), Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTPN22), and C-C Motif Chemokine Receptor 6 (CCR6) [40, 63]. Moreover, studies performed aiming to confirm the role of T cells showed that T cell deficient mice are protected from arthritis, and clinical trials performed to inhibit effector T cells activities demonstrate the improvement of clinical symptoms [46, 90, 91]. IL-17A, one of the cytokines secreted by Th17 CD4<sup>+</sup> T cells, amplifies local inflammation and the production of TNF-α and IL-6, which in turn promote RANKL expression by induction of an intense osteoclastogenesis [37]. Th17 CD4<sup>+</sup> T cells also express RANKL, but this molecule only stimulates an additive effect and is not sufficient to induce osteoclastogenesis, independently, in this disease scenario [37, 43, 46]. It was also reported that these cells stimulate the recruitment of OCs progenitors via increasing chemokine production by BM MSCs [40]. Recently, it was shown that IL-22, produced by the Th22 CD4<sup>+</sup> T cells, promotes osteoclastogenesis and enhances bone destruction in arthritic mice [46, 92]. Disease severity is shown to be markedly reduced in collagen-induced arthritic mice deficient in IL-22 [92], and elevated IL-22 in serum is also associated with disease activity in patients with RA [92].

Interestingly, it has been found that a particular type of Th17 CD4<sup>+</sup> T cells, derived from FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells (called exFoxP3 Th17 T cells), have a much stronger pro-osteoclastogenic activity than conventional Th17 CD4<sup>+</sup> T cells [86, 93]. Under arthritic conditions induced in mice model, FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells lose FoxP3 by the action of IL-6 produced by synovial fibroblasts [46, 83, 94]. Indeed, FoxP3<sup>+</sup> IL-17<sup>+</sup> CD4<sup>+</sup> T cells—a transition state during the conversion to exFoxP3 Th17 T cells—are frequently observed in synovial tissues of patients with active RA, as compared with those with inactive RA, suggesting a pathogenic role for this subset in this pathological condition [46, 95]. Equally important is the fact that Foxp3<sup>+</sup> IL-17<sup>+</sup> CD4<sup>+</sup> T cells were also observed in periodontal tissues of patients with severe periodontal disease [96, 97]. Notably, in a ligature-induced periodontitis mouse model, it was recently shown that Th17 CD4<sup>+</sup> T cells eradicate the bacteria while also inducing bone degradation and tooth loss, which is crucial for the termination of oral infection, avoiding bacterial systemic dissemination [98]. Taken together, it was concluded that Th17 CD4<sup>+</sup> T cells orchestrate the host defense against oral microbiota by regulating both osteoclastic bone resorption and antimicrobial immunity [98].

It was reported that IL-4 produced by Th2 T cells inhibits OCs formation and function *in vitro* [86, 99, 100]; nonetheless, no functional activity has been reported *in vivo*. On the other hand, Th1 CD4+ T cells, which counter regulate Th2 cells, are found in the synovium fluid of patients with active RA [101], although it has been demonstrated that the secretion of IFN-γ by this T cell subset strongly inhibits osteoclastogenesis and protects against bone tissue degradation by OCs [102]. IFN-γ induces a strong inhibition of the RANKL-induced activation of the NF-κB, via a

rapid degradation of TRAF6 [102]. In arthritic synovium, Th1 CD4+ T cells are not considered to be activated but often display an exhausted phenotype and express low levels of IFN-γ [86–89].

It is already known that FoxP3<sup>+</sup> Treg CD4<sup>+</sup> cells play an indispensable role in maintaining immune homeostasis, but also exert a strict anti-osteoclastogenic activity [68, 103–106]. In rheumatic patients, the number of FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells is inversely related to osteoclastogenic markers and disease severity [68, 105, 107]. These results accompany findings in which mice deficient in FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells were prone to arthritis, showing joint destruction and generalized bone loss, supported by higher number of OCs in joints [105]. The reintroduction of FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells into these mice significantly reduced arthritic clinical symptoms [105]. As discussed in previous section, OCs express the co-stimulatory molecules CD80 and CD86, and osteoclastogenesis can be regulated via CTLA-4, promoting OCs apoptosis, and thus suppressing bone destruction [81]. Notably, BM resident FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells express higher levels of CTLA-4, than peripheral FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells [82]. These resident FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells remove CD80/CD86 from the surface of OCs precursor cells by CTLA-4 mediated trans-endocytosis, potentially leading to reduced co-stimulation by OCs [82]. Therefore, the interaction between OCs expressing CD80/CD86 and FoxP3<sup>+</sup> Treg CD4<sup>+</sup> T cells expressing CTLA-4 is suggested as important player for the cross talk between these cells to support bone homeostasis [81, 82].

More recently, the term immunoporosis—a subarea under osteoimmunology' umbrella—was proposed for the field that studies the importance of immune system for osteoporosis establishment [108]. Osteoporosis—defined by a loss of bone mass and microarchitecture, has a multifactorial etiology but endocrine factors such as hyperparathyroidism, vitamin D deficiency, and menopause are primarily implicated [109]. The disease stems mainly from the cessation of ovarian function, where declining estrogen levels result in the stimulation of bone resorption, leading to a period of rapid bone loss [109]. At the cellular level, the central mechanism by which sex steroid deficiency induces bone loss is via an increase in OC formation and life span [110].

Estrogen exhibits the potential to stimulate the differentiation and survival of regulatory T cells, which in turn suppress the expression of proinflammatory cytokines from Th17 T cells and inhibit bone resorption. In addition, many genetic and non-genetic factors intensify the negative impact of estrogen deficiency on the skeleton, including gut microbiota profile [109]. Indeed, sex steroid deficiency increases gut permeability, allowing intestinal microbiota to activate and expand Th17 and TNF-α<sup>+</sup> T cells [111]. These expanded T cells increase S1PR1 (sphingosine-1-phosphate receptor 1) expression, which promotes their egress from intestine and influx into BM through CXCR3 and CCL20-mediated mechanisms [111, 112]. Additionally, this steroid deficiency-associated bone loss was prevented by probiotics administration [111, 112]. In this regard, several studies demonstrated that *Lactobacillus species* alleviates gut inflammation and improved barrier function of intestine [113]. Moreover, it was shown that *Lactobacillus rhamnosus* administration enhances bone mass in eugonadal mice [112, 114, 115], inhibits osteoclastogenesis, and skews balance of Th17 T cells to regulatory T cells, under *in vitro* and *in vivo* conditions [112, 115]. Collectively, these studies highlight the osteoprotective role of this probiotic, thereby opening novel avenues in the management and treatment of postmenopausal osteoporosis.

### *Perspective Chapter: Breast-Tumor-Derived Bone Pre-Metastatic Disease – Interplay... DOI: http://dx.doi.org/10.5772/intechopen.107278*

Finally, the effect of B cells in bone is more bidirectional, as compared with T cells, as B cells require the endosteal BM surface to their ontogenesis [78]. Indeed, B cell transcription and growth factors that control B cell differentiation play important roles in bone homeostasis, indicating the tight interaction between this immune cell lineage and bone [116]. In RA, the autoimmune process starts with the presentation of auto-Ags to CD4<sup>+</sup> Th T cells, which help B cells to differentiate into plasma cells that produce auto-Abs, such as rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP), trademarks of this disease [106, 117, 118]. Of note, the substantial number of Tfh cells in the synovial tissue correlates with disease severity [119, 120]. Auto Abs and immune complexes promote bone erosion through FcRγ signaling in OC precursor cells or innate immune cells [121, 122]. More recently, it was demonstrated that plasma B cell numbers increased in BM region near the inflammatory joints during arthritis [119], due to enrichment of plasma B cells survival factors such as IL-6, BAFF, and APRIL [119]. Locally, plasma B cells provide RANKL, TNF-α, IL-17, and Ab-mediated costimulatory signals that cooperate to powerfully promote osteoclastogenesis [119]. Genetic ablation of RANKL in B cells resulted in amelioration of periarticular bone loss, but not of articular erosion or systemic bone loss, in RA [123], and was slightly but significantly protective of ovariectomy-induced bone loss [124].

After reviewing the progress on the central roles of adaptive immunity in the establishment of some bone disorders, we will now explore the knowledge behind the participation of tumor-primed T and B cells in the development of bone pre-metastatic niche, which will lead in turn to the establishment of breast-cancer-derived bone metastases.
