T-helper cells in the etiopathogenesis of periodontal disease: A mini review.

Our traditional understanding of the T-helper (Th)1/Th2 paradigm in periodontal disease has undergone considerable changes in recent years. This review focuses on the Th subsets, including the recently identified cells of the CD4 lineage, their activation pathways and effector function in periodontal disease. The roles of Th17 and regulatory T (Treg) cells in disease pathogenesis have been explored. Newer Th subsets such as Th9 and Th22 cells and their potential role in periodontal disease have also been outlined.

INTRODUCTION

The central role played by T cells in the etiopathogenesis of periodontal disease was recognized following the seminal work of Ivanher and Leanyi.[1] However, with a greater understanding of their biological functions, T cells are now believed to be involved in the homeostasis of periodontal tissues,[2] modulation of the inflammatory/immune responses[3] and mediation of the bone loss observed in periodontal disease.[4]

T cells are classified based on their function into various categories such as helper T (Th) cells, cytotoxic T (Tc) cells and regulatory T (Treg) cells. This article will review our current understanding of the activation and effector functions of the Th (CD4) cells, including the recently identified subsets; and their potential role in periodontal disease.

Th cells were so designated because they played a crucial role in the process of antibody production by the B cells.[5] Originally, the presence of two different subsets of Th cells-Th1 and Th2 was described by Parish and Liew.[6] Later, Mossman and Coffman[7] delineated two distinct cytokine profiles associated with the Th1 and Th2 cells. Ever since, the Th1/Th2 paradigm has been used to explain the pathogenic mechanisms involved in several inflammatory/immune disorders including periodontal disease.[8] Seymour and coworkers[9] have suggested that the early stable periodontal lesion was associated with a robust infiltration of Th1 cells. On the contrary, other authors have proposed that a Th2 cytokine profile was associated with a stable lesion, while the progressive lesion was characterized by the Th1 profile.[1011] In recent years, various mechanisms such as the nature of the antigen, dendritic cell (DC) profile, the cytokine milieu in the tissues have all been shown to regulate the Th1/Th2 response in periodontal disease.[1213]

Th cells have been identified to be a more heterogeneous lot than previously appreciated. Th17[14] and Treg cells[15] were added to the Th lineage and more recently, other subsets such as Th22[16] and Th9 cells[1718] have been identified that were phenotypically distinct from the existing Th cells. Further, it has been shown that Th cells demonstrate plasticity, that is, the ability to transform from one subset to the other.[1920] Clearly, the traditional Th1/Th2 paradigm is an oversimplification of the complex Th mechanisms that are involved in the pathogenesis of periodontal disease.

TH1 CELLS

Th1 cells are believed to have evolved to afford protection against the intracellular pathogens. These cells produce the interferons (IFN) that are involved in macrophage activation, which in turn plays an important role in phagocytosis, complement fixation and opsonization.[21]

Development

Th1 development requires inductive signals that initiate a network of intercommunicating intracellular events that culminates in the differentiation of the naive T cell to a differentiated Th1 cell.

Inductive signals

Th1 polarization is induced by the cytokines interleukin (IL)-12 and IFN-α, both of which are present in the inflammatory milieu. These cytokines are normally produced by the DCs or the natural killer (NK) cells.[22] In the periodontal environment, DC infiltration has been strongly associated with gingival inflammation.[23] These cells have toll-like receptors (TLR) on their surface that enable them to respond to the pathogen-associated molecular patterns-lipopolysaccharide, fimbriae, etc,of the periodontopathogens. NK cells have also been identified in the inflamed periodontium and are thought to respond to a variety of stimuli, including lipid antigens such as lipotechoic acid, in a TLR independent, CD1d-dependant manner.[24]

Intracellular signaling pathway

IFN-α/IFN-γ in conjunction with IL-12 activates signal transducer and activator of transcription-1 (STAT-1) in the naive CD4 T cells. Activated STAT-1 upregulates the master regulator of Th1 differentiation-T box expressed in T cell (T-bet). Activation of T-bet leads to induction of the receptor IL-12Rβ2, making these cells more responsive to IL-12. This in turn, causes activation of STAT-4, which then upregulates IFN-γ production by the Th1 cells. At the later stages of differentiation, IL-18Rα receptor expression is upregulated, thereby, making these cells responsive to IL-18. Concomitant action of IL-12 and IL-18 leads to the full repertoire of IFN-γ production that is characteristic of the effector function of Th1 cells.[2526]

TH2 CELLS

Th2 cells are thought to have evolved as protection against parasitic helminths. These cells produce IL-4 in addition to IL-5 and IL-13; cytokines that are involved in immunoglobulin (Ig) class switching in B cells.[2127] The importance of Ig-G that is produced as a result of this class switching in periodontal disease pathogenesis has been well-documented.[28]

Th2 development is induced by production of IL-4, which in turn is produced by the naive T cells or the mast cells/macrophages .[2930] In the periodontal milieu, naive T cells constitute only a small proportion of T-cell infiltration within the tissues; it is comprised predominantly of the memory or the effector cells.[31] Therefore, they are unlikely to be the major source of IL-4 in the gingiva. On the other hand, both macrophages and mast cells have been identified in the periodontal environment and as early responders, they are the likely source of IL-4 production.[832]

Intracellular signaling

IL-4 signaling results in activation of STAT-6 which then upregulates the master switch of Th2 differentiation, GATA-binding protein-3 (GATA-3).[33] In addition to GATA-3, STAT- 5 signaling is also required for complete Th2 differentiation.[34] GATA-3 auto-amplifies its own gene, subsequent activation is, therefore, not dependant on other signals. Consequently, Th2 cells are thought to be a more stable phenotype.

TH1/TH2 RECIPROCITY

The two Th1/Th2 subsets exert a suppressive effect on each other through a variety of mechanisms. Their signature cytokines, IL-4 and IFN-γ suppress each other, i.e., IFN suppress Th2 responses while IL-4 production downregulates Th1 responses. The intracellular signals are also capable of similar responses. For example, GATA-3 suppresses STAT-4 and thereby Th1 responses, while STAT-5 reduces T-bet expression. Tbet by itself, can downregulate GATA- 3 expression.[3536]

TH1/TH2 CELLS IN PERIODONTAL DISEASE

Several studies have discussed the role of Th1 and Th2 cells and their cytokine profiles in periodontal disease. As detailed descriptions are available elsewhere,[37] they will be dealt with only briefly here. Over the past decade, it has been proposed that as stable periodontal lesions resemble a delayed type hypersensitivity lesion and progressive lesion involves large number of B cells, these lesions may be mediated by Th1 and Th2 cells, respectively. It has been shown that a strong innate response leads to a Th1 response under the influence of IL-12, IL-2 and IFN-γ while a weak innate response leads to a Th2 response under the influence of IL-4 cytokines. In a stable lesion, IFN-γ enhances the phagocytic activity of both neutrophils and macrophages and hence contains the infection.[38] In case of a poor innate immune response and minimal Il-12 production, a weak Th1 response may not contain infection. Mast cell stimulation and the subsequent production of IL-4 would encourage a Th2 response, B-cell activation and antibody production. If these antibodies are protective and clear infection, the disease will not progress but if they are not protective, as in case of IgG2, the lesion will persist.[28] Continued B-cell activation may result in large amounts of IL-1 and hence tissue destruction.[39-41]

Despite its simplicity, the Th1/Th2 model does not adequately explain many findings with respect to T-cell-mediated immune responses. Various studies have reported discrepancies with regard to predominance of Th1 or Th2 response or the involvement of both Th1 and Th2 cells in diseased tissue.[10-13]

Another subset of CD4+ T cells that explains some of the deficiencies in the classic Th1/Th2 model, have been termed “Th17 cells”.[42]

TH17 CELLS

For several decades, the Th1/Th2 paradigm held sway and was used to describe the progression of inflammatory and autoimmune disorders. Although IL-17 was known for a long time, it was thought to be produced mainly by the Th1/Th0 cells. Th-17 cells were identified as a separate phenotype only recently.[43] The signature cytokine of these cells is IL-17A, but they are also known to produce IL-21 and IL- 22. IL- 17A is thought to be important for the recruitment of neutrophils as they up-regulate CXCL8 expression. It is thus, believed that Th17 cells have evolved primarily to provide inflammatory protection against the extracellular pathogens. They are also believed to be involved in inflammatory bone disorders such as rheumatoid arthritis.[44]

Inducing signals

However, Th17 cells are thought to develop in the presence of transforming growth factor-β (TGF-β) in synergy with IL-6 and/or IL-1. In addition, IL-21 and IL-23 signals are necessary for expansion and stabilisation of their phenotype.[4546] Of these inductive signals, IL- 23 (a member of the IL-12 family) has been identified in the inflamed tissues as part of the early chemokine/cytokine infiltrate.[47] The presence of IL-6, IL-1 and TGF-β in the chronically inflamed gingival tissues is well-documented and would seem to create an environment conducive to Th17 development.[48]

Intracellular pathway

As far as Th17 development is concerned, the intracellular pathways have not been as well-defined as the inductive signals. It is, however, generally agreed that the master regulator of Th17 development is retinoic acid-related orphan receptor γt (RORγt). It is also understood that STAT-3 signaling is critical for the development of the Th17 cells. It is not yet fully understood if these signals are interdependent on each other. Thus, the intracellular signaling pathway for Th17 development, stabilization and terminal differentiation is yet to be fully understood.[49]

TH17 CELLS IN PERIODONTAL DISEASE

Th17 cells have been identified in the periodontal tissues.[50-52] There is, however, a considerable difference between the murine and human cell types. Th17 has been proposed to exert both proinflammatory and anti-inflammatory functions.[53] It was demonstrated that Porphyromonas gingivalis outer membrane protein induced a significant increase in the production of IL-17.[54] IL-17 has been shown to stimulate epithelial, endothelial and fibroblastic cells to produce IL-6, IL-8 and PGE2.[55] In addition, IL-17 induces receptor activator of nuclear factor-κ B ligand (RANKL) production by osteoblasts[5657] and thus influence osteoclastic bone resorption. Although there are conflicting reports, several authors have identified IL-17 in the gingival crevicular fluid.[505158]

It has been hypothesized that Th17 cells may be involved in Th1 modulation and enhanced inflammatory mediators’ production by gingival fibroblasts in periodontal disease. Further, it has also been proposed that Th17 cells may be primary source of RANKL production in periodontal disease.[50]

REGULATORY T (TREG) CELLS

Another set of CD4+ T cells that were identified have a function that is unlike that of the ones described previously. These cells have a regulatory function in that they are known to suppress T-cell proliferation and activation.

Regulatory cells were identified as a separate cell by Gershon[59] and called suppressor cell. It was only recently that they have been delineated as a fully functional CD4+ T cell.[60] These cells secrete TGF-β and importantly IL-10 and are, therefore, required for regulation of inflammatory responses. They play a critical role in tolerogenic responses and suppression of inflammatory responses against self-antigens. Regulatory T cells are classified into natural (Treg) and adaptive cells (Tr1, Th3). While, Treg cells have evolved to provide protection against autoantigens, Tr1 cells are required for tolerogenecity.[61]

Inducing signal

Treg cells are thought to develop in the presence of TGF-β, but in the absence of IL-6, IL-1 and this process is favored by IL-2 and retinoic acid.[62]

TGF-β leads to activation of forkhead transcription factor (Foxp3) which is the master regulator of Treg cells. It has been postulated that Foxp3 activation may be different in natural and adaptive Treg cells. The intracellular signaling mechanisms involved in Treg development are yet to be fully elucidated.[6263]

Mechanism of Immune-Suppression by Treg Cell

Treg cells are now widely regarded as the primary mediators of peripheral tolerance. Peripheral tolerance is the lack of responsiveness of mature lymphocytes in the periphery to specific, weak antigens. The various potential suppression mechanisms used by Treg cells can be grouped into four basic modes of action:[61]

Suppression by inhibitory cytokines:

Inhibitory cytokines, such as IL-10 and TGF-β play important roles in Treg cell-induced suppression, but, these cytokines may not be essential for Treg-cell function.[64]

Suppression by cytolysis:

Treg-cell-cytolytic activity is mediated by granzyme A and perforin through the adhesion of CD18, resulting in suppression of B cell, cytotoxic T cells and NK cell function. Moreover, activated Treg cells induce apoptosis of effector T cells through a TRAIL-DR5 (tumor necrosis factor-related apoptosis-inducing ligand-death receptor 5)-mediated pathway.[6566]

Suppression by metabolic disruption:

Another mechanism by which Treg cells mediate their suppressive effect is termed as ‘metabolic disruption’ of the effector T-cell target. The expression of high levels of CD25 by Treg-cell is thought to consume available IL- 2 and therefore starving actively dividing effector T cells.[67]

Suppression by modulation of DC maturation or function:

Treg cells also suppress immune response by targeting DCs. In addition to the direct effect of Treg cells on T-cell function, Treg cells might also modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Direct interactions between Treg cells and DCs through the costimulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA4), attenuates effector T-cell activation. Treg cells condition DCs to express indoleamine 2,3-dioxygenase (IDO), a potent regulatory molecule, which is known to induce the production of proapoptotic metabolites from the catabolism of tryptophan. This results in suppression of T-cell proliferation. In addition to inducing DCs to produce immunosuppressive molecules, Treg cells may regulate the capacity of DCs to activate effector T cells through downregulation of CD80 and CD86 molecules.[6869]

Treg cells can also influence immune responses by modulating the recruitment and function of other cell types such as mast cells, monocytes and macrophages.[70]

TREGS CELLS IN PERIODONTAL DISEASE

Evidence regarding the exact role played by Treg cell in mediating periodontal disease continues to be controversial. Nakajima[71] and Cardoson[72] demonstrated that periodontitis patients showed an increased percentage of Treg cells in gingival connective tissue compared to gingivitis patients. It was concluded that Tregs levels were upregulated in chronic periodontitis lesions as protection against self-antigens such as collagen-1. Ernst[73] demonstrated that Treg cells downregulated RANKL expression in periodontal disease. However, their capacity to provide immunoregulatory function in periodontal disease has been questioned. Dutzan[74] suggested that Treg cells may not have a regulatory function due to lack of CTLA-4 expression that is required for cell-cell contact inhibition of T-cell proliferation. Further, Okui[75] suggested that Treg cells in gingival tissues may not have similar functions as that in circulation due to differences in Foxp3 expression in the two.

To summarize, although the exact nature of the role (protective vs. non-protective) played by Treg cells is not known, these cells exert important influences on the overall T-cell response.

TH17-TREG CELL RECIPROCITY

As, in the case of Th1/Th2 profile, counterbalancing mechanisms in the local environment contribute to the overall Treg/Th17 responses. TGF-β determines the differentiation of both Th17 and Treg cells. Activated CD4 T cell express both RORγt and Foxp3, but RORγt function is antagonized by Foxp3. In the presence of proinflammatory cytokines such as IL-1, IL-6 and low concentration of TGF-β, RORγt is further unregulated, whereas Foxp3 expression is inhibited. On the other hand, in the absence of these proinflammatory cytokines, high concentration of TGF-β favor Foxp3 expression and result in Treg-cell differentiation. In inflammatory states, retinoic acid levels are suppressed, as a result of which Treg differentiation is downregulated, thus tipping the balance in favor of a Th17 response.[63]

It could thus, be hypothesized that in a progressive periodontal lesion that is dominated by excessive proinflammatory cytokine production, Th17 cell would predominate. Conversely, a stable lesion, characterized by matrix formation and TGF-β production, would be predominantly associated with Treg cells. However, experimental evidence for such a phenomenon is yet to be obtained.

TH22 CELLS

Th22 cells are characterized by production of IL-22 (produced by Th17 cells as well) but low production of IL-17 and IFN-γ.[76] These cells are localized in the epithelium and are involved in the production of antimicrobial peptides like defensins.[77] These cells have been identified in dermal diseases,[7778] but their role in periodontal disease has not yet been determined. However, considering the high expression of the β-defensins in gingival epithelium, it seems to be reasonable to assume that these cells are involved in the immune mechanisms of the gingiva.

The differentiation pathways involved in the development of these cells are yet to be fully elucidated but it is known to be induced by aryl hydrocarbon receptor (AHR) and RORc, and primed by high levels of IL-6 and TNF-α.[78]

TH9 CELLS

IL-9, first described as a Th2 cytokine[79], was shown to be produced by a distinct subset of Th cells termed Th9 cells, when they were exposed to TGF-β and IL-4.[1718] IFN-γ, on the other hand suppresses the activation of Th9 cells.[80] Although they have been classified as a separate Th cell subset, a few authors have questioned their existence.[81]

IL-9 is involved in early hematopoietic ontogeny, can influence/affect proinflammatory cytokines such as TNF, IL-1, IL-17 and IFN-γ[82] and also affect differentiation and functioning of both Th17 and Treg cells.[83] IL-9-mediated signaling responses have been shown to be dependent on the Janus kinase (JAK-1)-signal transducer and activator of transcription (STAT-1,3,5) pathways.[84] The transcription factors necessary for the differentiation of human Th9 cells is not yet known. Their role in periodontal disease pathogenesis is not yet recognized.

Table 1 and Figure 1 summarizes the activation and function of various Th cell subsets.

Table 1

Category, differentiation and function of effector CD4+ Th cell

Figure 1

Diversification of CD4 T cell lineages

CONCLUSION

The pathogenic mechanisms governing periodontal disease is complex and results from disruption of a delicate balance that exists between the various Th cells and their effector cytokines. This review has highlighted some of the intracellular mechanisms involved in Th-cell activation. Even as this manuscript was under preparation, other Th cells have been proposed to exist, with widely differing functions. Whether these cells are actually different cell lineages or a member of a large T-cell family that have not yet attained terminal differentiation, is yet to be fully ascertained. Further investigations are required before we fully appreciate the role of Th cells in periodontal disease. In any case, the simplistic version involving the Th1/Th2 cells alone, no longer retains validity.