2.3.2. B Lymphocytes

The characterisation of B cell subsets in gingival tissues was recently described by Mahanonda et al. [39]. The authors reported very few naïve B cells (<8%) in all stages of healthy and diseased tissues. Additionally, memory B cells (CD19<sup>+</sup>CD27<sup>+</sup>CD38−) represented the majority of the B cell population in the clinically healthy gingiva and were detected in the connective tissue subjacent to the apical region of the junctional epithelium, which could be due to the local low-grade inflammatory response to a constant challenge of the biofilm. The authors highlighted the importance of detecting memory B cells in clinically healthy human gingiva since very little is known about memory B cells residing in human nonlymphoid tissues. The minimal presence of B cells in healthy gingiva was also reported by others [6,7,35,40]. Such low levels might be important to avoid bone loss around teeth due to the subclinical inflammation that occurs in the clinically healthy periodontium.

Another aspect of the B cell biology that is relevant for gingival homeostasis is the production of antibodies against periodontal pathogens, which can contribute to host protection [41,42]. Page et al. [43] demonstrated that immunisation using *P. gingivalis* as antigen could reduce the onset and progression of alveolar bone loss in non-human primates. Also, Shelburne et al. [41] suggested that anti-*P*. *gingivalis* HtpG antibodies predict health in patients susceptible to periodontal disease. The potential role of B cell humoral immunity in maintaining homeostasis needs further investigations. A description of the main functions of T and B cells' subsets in the periodontal tissues is presented in the Table 1.

### *2.4. Changes with Age*

Furthermore, age is also a variable that needs to be considered when evaluating the lymphocyte function in healthy periodontium. The effects of aging on periodontal tissues are thought to intensify alveolar bone resorption in elderly individuals [44]. Witkowski et al. [45] reviewed the proteodynamics in aging human T cells and reported that the proteolytic elimination of altered proteins, as well as modulation of the activity of those remaining, leads to the dynamic change of proteome composition and function in aging lymphocytes. Ebersole et al. [44] reported that several B cell and plasmacyte genes are altered in aging healthy gingival tissues, which are mainly associated with antigen-dependent activation and B cell differentiation/maturation processes. Aging T and B cell dynamics requires further comprehensive analysis and may influence the pathophysiology of periodontal disease.

### *2.5. T and B Lymphocytes in Periodontal Inflammation*

In 1983, Okada et al. [46] published a very elegant paper characterising the immunocompetent cells on histological sections from diseased human gingiva. According to the authors, human periodontitis contains numerous sets of infiltrating cells which are organized unusually, with a region rich in T lymphocytes and monocytes/macrophages just subjacent to the pocket or sulcular epithelium; and a region in the central lamina propria, located farther away from the microbial agent, which is rich in B cells and plasma cells and poor in T lymphocytes. Furthermore, the same group characterised the T lymphocyte subsets (T4+ and T8+) in the inflamed gingiva from human periodontitis and showed the ratio T4+/T8+ was lower in gingival tissue than in peripheral blood [47]. Dutzan et al. [6] evaluated the major cell subsets and revealed that the lymphocytic compartment, CD3+T cells remained the dominant population in both health and disease, ye<sup>t</sup> in disease the total number of T cells is much greater, reflecting a 10 fold increase in total inflammatory cells [6].

The role of T cells in the immune dysregulation of periodontitis has been consistently revised by Campbell et al. [1]. Activated Th1, Th2, and Th17 cells can produce a variety of pro-inflammatory cytokines, such as IL-1β, IL-17E (IL-25) and IL-17, that activate other immune cells such as dendritic cells, neutrophils, and B cells. Activation of both T cells and subsequently, B cells can cause the production of the receptor activator of nuclear factor κ B -Ligand (RANKL), which leads to alveolar bone resorption by osteoclasts, resulting in tooth loss. Moreover, the activation of B cells by Tfh in either peripheral lymph nodes or tertiary lymph organs can result in clonal activation of B cells, which produce antibodies to recognise bacterial components; however, production of autoantibodies to collagen, fibronectin and laminin can contribute to local destruction of the gingival tissue. Finally, a lack of Treg cells or an inability of those present to reduce local inflammatory responses by other immune cells may play a role in the chronic inflammation associated with periodontitis [1].

**Table 1.** A summary of the main functions of mentioned T and B cells in periodontal health and disease.


Treg: regulatory T cells; MAIT cells: mucosal-associated invariant T cells; Th: helper T cells; CD: cluster of differentiation; SOFAT: secreted osteoclastogenic factor of activated T cells; Tfh: T-follicular helper; Breg: regulatory B cel.

T cells have a crucial role in the tissue secretion of IL-17, a cytokine strongly associated with bone loss around teeth. Chen et al. [48] reported that IL-17 and IFN-γ levels in biopsy specimens of gingival lesions from chronic periodontitis patients were higher than those in the healthy controls. Moreover, relative IFN-γ, IL-17A, and T-bet mRNA levels were also significantly higher in patients with chronic periodontitis compared to controls, suggesting that Th17 and Th1 cells might be involved in the pathogenesis of chronic periodontitis. Dutzan et al. [6,35] characterised IL-17-secreting cells within the hematopoietic compartment in healthy and periodontitis gingival samples and found a significant increase in IL-17<sup>+</sup> cells in diseased sites. The major source of IL-17 was CD4+ T cells, with minimal contribution from CD8, γδT, and non-T-cell sources.

Moreover, the percentage of CD4+ T cells producing IL-17 significantly increased in periodontitis. CD4+ T cells preferentially upregulated IL-17 and not IFN-γ in gingival tissue from periodontitis patients. The same group also reported that Th17 cells in periodontitis are dependent on the local dysbiotic microbiota, and both IL-6 and IL-23 are required for their accumulation. Also, pharmacologically targeting RORγt, a transcription factor relevant for Th17 di fferentiation, reduces alveolar bone loss in a murine model of periodontitis [49]. On the other hand, Parachuru et al. [4] presented an interesting paper that compared healthy/gingivitis tissues with chronic periodontitis tissues. Among other goals, they aimed to determine the identity of FoxP3 and IL-17A positive cells in periodontal tissues. The authors reported that Th17 cells either do not exist in periodontal disease or are present in small numbers and that, as with other chronic inflammatory lesions, the source of the relatively small amounts of IL-17 may be mast cells. Moreover, they also suggested that Tregs may have an important role in the pathogenesis of the chronic inflammatory periodontal disease. Such a statement needs to be better investigated since it might a ffect our present understanding of the Th17/Treg imbalance that leads to periodontal disease progression.

Besides the importance of IL-17 secreted by CD4+ T cell or mast cells, a novel T cell-secreted cytokine, called secreted osteoclastogenic factor of activated T cells (SOFAT), that can induce osteoclastogenesis in a RANKL-independent manner, has been described in periodontal tissues [50]. The authors showed that the mRNA and protein levels of SOFAT were significantly higher in the gingival tissue of periodontitis patients compared to controls. More recently, Jarry et al. [51] demonstrated that B-lineage cells, including plasma cells, also exhibited strong staining for SOFAT in diseased periodontal tissue. Therefore, SOFAT might have an important role in periodontal disease by activating RANKL related osteoclastogenesis.

The characterisation and identification of interstitial T cells are relevant to understanding the immunopathogenesis of periodontitis. Bittner-Eddy et al. [52] have made an important contribution to this subject by describing a flow cytometry assay that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. They reported that, unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44hi). The authors reported the presence of antigen-experienced *P. gingivalis*-specific CD4 T cells in nasal-associated lymphoid tissues following oral feeding of mice with *P. gingivalis.* Such di fferentiation might be critical for future understanding of the players driving alveolar bone destruction.

B cells infiltrate and dominate sites showing progressive chronic inflammatory periodontal disease in humans [53]. It has been shown that periodontitis lesions contain significant numbers of immunoglobulin-bearing lymphocytes and plasma cells, suggesting that the clinical progression of the periodontal lesion is followed by a shift in cellular infiltrates from predominantly immunoglobulin-negative lymphocytes to IgG and IgM-bearing lymphocytes and plasma cells [54]. Oliver-Bell et al. [55] demonstrated that B cells make a substantial contribution to alveolar bone loss in murine periodontitis, probably due to B-cell activation and expression of RANKL in the gingiva. Abe et al. [56] reported that ligature-induced periodontitis resulted in significantly less bone loss in B cell-deficient mice compared with wild-type controls, supporting the importance of B cells in periodontal bone loss. The authors also suggested that two cytokines of the TNF ligand superfamily, a proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), might be potential therapeutic targets in periodontitis [56].

Mahanonda et al. [39] have characterised B cell subsets in gingivitis and periodontitis. The density of memory B cells in periodontitis lesions was significantly lower than in healthy and gingivitis tissues. On the other hand, Ab-secreting cells were the major cell type in the CD19+ B cell population, with the mean percentage of Ab-secreting cells being significantly higher than that of memory B cells. Moreover, an abundance of CD138+ plasma cells was observed in periodontitis tissues. The authors reported that plasma cells were arranged in clusters detected at the base of the periodontal pocket area and scattered throughout the gingival connective tissue, especially apically toward the advancing front of the lesion [39].

B cells in patients with periodontal disease may contribute to chronic systemic inflammation through constitutive secretion of IL-8 and IL-1β [8], but the in situ impact of such cytokine production should be elucidated. Kawai et al. [57] have demonstrated that B cells can be the cellular source of RANKL for bone resorption in homogenised gingival tissue from sites showing periodontal disease. Moreover, Malcolm et al. [58] have shown that the percentage of B cells expressing RANKL was elevated following *P. gingivalis* infection in gingival tissues. Oliver-Bell et al. [55] have also investigated the impact of *P. gingivalis* infection in the RANKL expression of B cells, showing that mice infected with *P. gingivalis* presented a significant increase in B-cell RANKL expression in the gingiva. Moreover, B-cell-deficient mice did not show *P*. *gingivalis*-induced alveolar bone loss. Recently, Kanzaki et al. [59] demonstrated that sRANKL and TNF-α cleaved from activated tumour necrosis factor-α-converting enzyme-bearing B cells might be important as an osteoclastogenic factor in periodontitis lesions.

Han et al. [10] suggested that B cells affect alveolar bone homeostasis in a murine model of periodontitis through antibody-independent and RANKL-dependent mechanisms. They reported that gingival memory B cells promote osteoclastogenesis and that this potential was increased by the development of periodontitis. Demoersman et al. [60] reported that a significantly higher percentage of CD27+ memory B cells was observed in patients with severe periodontitis. At the same time, human B1 cells, which were previously associated with a regulatory function, decreased in such patients. The authors also reported that the RANKL expression increased in every B cell subset from severe periodontitis patients and was significantly greater in activated B cells than in the subjects without periodontitis. Moreover, an interesting literature review published by Zouali [54] supports that B cells are key participants in RANKL-mediated bone resorption. Activated RANKL-positive B cells can exacerbate alveolar bone loss in a RANKL-dependent manner in animal models. On the other hand, blocking RANKL, B-cell-activating factor (BAFF), and a proliferation-inducing ligand (APRIL) reduces alveolar bone loss in experimental models of periodontitis. Coat et al. [61] reported that periodontal parameters could be significantly improved after treatment with rituximab, concluding that anti-B lymphocyte therapy could be beneficial in improving de clinical conditions of patients with periodontitis.

Besides the fact that B cells positively activate immune responses, functioning as APCs and producing antibodies, regulatory B cells (Bregs) have been shown to exert a suppressive role in immune response [62]. B10 cells are a Breg cell subset that produces IL-10 and therefore, negatively regulates the inflammatory responses [63]. B10 cells are present in gingival tissues of patients with and without periodontal disease, but in significantly higher levels in periodontal disease lesions [5.89 ± 2.02) when compared to healthy tissues (0.1 ± 0.3, *p* < 0.01) [64]. Yu et al. [65] demonstrated that the local induction of IL-10 competency of B10 cells was associated with the inhibition of both inflammation and bone loss in ligature-induced experimental periodontitis. Wang et al. [66] also reported that the adoptive transfer of B10 cells significantly inhibited inflammation and bone loss in a mouse model of experimental periodontitis, suggesting a potential novel principle of treatment for periodontal diseases. It has been suggested that the in vitro treatment of B10 cells with a combination of IL-21, anti-Tim1, and CD40L might inhibit periodontal bone loss in ligature-induced experimental periodontitis [65]. A schematic of the lymphocyte subsets and their possible contribution to periodontal homeostasis and inflammation is presented in the Figure 1.

**Figure 1.** A summary of how mentioned T and B cells can contribute to periodontal health and disease. In periodontal health, Treg and CD8+ T cells contribute to periodontal homeostasis through the production of IL-10 and TGF-β. γδ T cells produce amphiregulin and IL-17 to promote periodontal homeostasis. B cells produce antibodies against periodontal pathogens, limiting the development of periodontal inflammation. In periodontal disease, activated Th1, Th2, and Th17 cells produce pro-inflammatory cytokines that contribute to tissue damage. Both T and B cells produce RANKL, which leads to osteoclast activation and alveolar bone resorption. Clonal activation of B cells by Tfh cells can lead to the production of autoantibodies to collagen, fibronectin and laminin, contributing to local tissue destruction. Lack of Treg cells or an impaired function probably impact on the development of periodontitis. IL-17 produced by other cells can also contribute to tissue damage via osteoclast activation. The figure was adapted from Lira-Junior & Figueredo [67].

The increased knowledge in T and B cells' biology, as well as their functions during gingival tissue homeostasis and inflammation might help in designing novel therapeutic strategies for bone disorders where these cells are a crucial part of the local tissue inflammation, such as periodontitis.
