CHAPTER 25 Molecular Biology of the Host-Microbe Interaction in Periodontal Diseases
This chapter provides an overview of the molecular biology of the host-parasite relationship from a cellular signalling perspective specific to pathogen-associated lipopolysaccharide (LPS). The implications of periodontal microbiota-associated LPS and other microbiota-associated molecular patterns (MAMPs) on the induction of the innate immune response, toll-like receptor (TLR) signalling, and periodontal pathogenesis are reviewed. The second half of the chapter highlights the pathobiology of periodontal disease and the induction of disease by proinflammatory cytokines that generate host-tissue destruction and alveolar bone loss. The chapter concludes with ramifications for the development of therapeutic strategies that perturb innate immune signal transduction for the treatment of periodontitis.
For the past two decades, the host response to the bacterial challenge originating from the dental biofilm has been considered to play a major role in initiation and tissue destruction of periodontal diseases.176 The significance of host-microbial interactions is reinforced by epidemiologic data indicating different susceptibilities to periodontal disease among individuals, in spite of the long-term presence of oral biofilm.20,21,194 Other studies demonstrating increased susceptibility and greater severity of periodontal disease in individuals with impaired immune response caused by systemic conditions also indicate the significance of the host response to the bacterial challenge.88,209 In the current paradigm of periodontal disease, periodontal pathogens are essential for disease initiation; however, the extent and severity of tissue destruction are largely dependent on the nature of the host-microbial interactions. These interactions are dynamic, since the microbial composition of the dental biofilm and host immune competency can vary widely between patients resulting in differences in host responses and subsequent alveolar bone loss. This concept has evolved in parallel with a more advanced appreciation of the immune response resulting in an increased emphasis focused on mechanisms of host-microbial interactions in periodontal disease pathobiology, as well as on the development of novel therapeutic strategies.
Periodontal diseases provide a unique situation to study microbial-host interactions. Over 500 different microbial species can be found in the oral biofilm;237 however, only a few of those are associated with periodontal disease.293,294 This implicates that the recognition of both nonpathogenic/commensal bacteria and pathogenic bacteria by the host requires vigilance and tolerance mechanisms to mount an appropriate response that can prevent dissemination of infection without inducing an exacerbated reaction that could result in damage to the host tissues. Direct recognition of microbes by the host is mediated by the recognition of MAMPs by pattern-recognition receptors (PRRs).35,37
Host response to periodontal infection requires expression of a number of bioactive agents, including proinflammatory and antiinflammatory cytokines, growth factors, and enzymes that are the result of the activation of multiple signalling pathways. This activation of intracellular signalling may initiate exclusively as an innate immune response associated with PRR-mediated sensing of MAMPs. However, since the oral cavity is one of the most readily colonized postnatal mucosal sites, the long-standing interactions between microbes (commensal and pathogenic) and host results in a “priming” of the immune system with the common presence of adaptive immune cells in the periodontal tissues.
The biologic mediators expressed as a result of PRR-signalling activation include costimulatory molecules involved in the induction of adaptive immunity.37 This results in a cascade of events that will establish very complex cytokine and signalling networks. There is accumulating evidence for a direct role of “classic” innate immunity signalling through PRRs in adaptive immune cells that can modulate the function of these cells. Abundant evidence indicates a key role for the adaptive immune response—humoral and cellular aspects—in mediating the host response to microorganisms existing within the oral biofilm and also in the majority of tissue destruction associated with periodontal diseases.* Even though cells participating in the adaptive immune response are considered to be the primary source of cytokines leading to bone resorption,168 additional data exist showing that periodontal bone loss occurs in the absence of B and T cells, suggesting a role for the innate immune response in periodontal disease initiation or progression.22,25,26
Innate immunity and inflammation are not synonymous; however, inflammation arises primarily in response to infection. More importantly, innate and adaptive immune responses are not mutually exclusive, and the bridge between these arbitrarily and didactically distinguished arms of the immune response has been shortened in recent years. However, considering that this chapter focuses on host-microbe interaction, we will emphasize the direct recognition of MAMPs by cells participating in the immune response and the molecular mechanisms activated downstream of this recognition (Figure 25-1).
Figure 25-1 Microbial-associated molecular pattern (MAMP) from microorganisms in the dental biofilm activate inflammatory responses in periodontal tissues. Biologic mediators will either affect neighboring cells (blue arrows) by inducing the expression of other mediators (e.g., RANKL) or by triggering chemotaxis (green arrows). Direct damage to periodontal tissues may also result after MAMPs stimulation (red arrows), such as metalloproteinase secretion by gingival and periodontal ligament fibroblasts. MAMP, Microbial-associated molecular patterns; RANKL, receptor activator of nuclear factor (NF) -κB ligand; KC, keratinocyte; Mac, macrophage; GF, gingival fibroblast; PDL, periodontal ligament fibroblast; OB, osteoblast; OC, osteoclast; EC, endothelial cell; PMN, polymorphonuclear neutrophil; Mon, monocyte; T cell, T lymphocyte, B cell, B lymphocyte.
Innate Immunity in Periodontal Diseases
Vigilance and Tolerance
Even though adaptive and innate immune responses are traditionally distinguished, in vivo they are integral parts of the host defenses against infections (i.e., innate immunity is not inactivated once the adaptive response is initiated). Innate immunity is required for the activation of a more specific adaptive immune response, but it also plays an important role in managing host-microbial interactions since the innate immune system is rapidly activated (within minutes) and is responsible for the defense during the initial hours and days of the infection, whereas adaptive immunity requires at least 7 to 10 days before an adequate cellular or humoral response occurs. This role of innate immunity is especially important considering that our host cells are outnumbered 10 : 1 by the microbial cells living on skin and mucosal surfaces. This coexistence, however, is usually pacific and even beneficial for one or both cohabitants but requires both vigilance and tolerance mechanisms that ultimately determine if the host response is to clear the infection, or suppression of the host response is required to maintain the homeostatic equilibrium between host and microbes.
With regard to periodontal diseases, current paradigm indicates that some groups or complexes of bacteria are more strongly associated with the presence of periodontal diseases, but this association is not universally found, as there are individuals harboring the disease-associated complexes that are free of periodontal disease. Moreover, the microbial flora of the oral cavity can include more than 500 different species and there is no uniformity on the level of infection that is sufficient to produce disease. The complexity of the oral microbial flora is particularly intriguing given the limited number of receptors that are able to recognize microbial antigens. Tolerance mechanisms probably play a role in modulating the host responses to commensal/nonpathogenic bacteria. One of the primary challenges of the innate immune system is to discriminate among a large number of periodontal pathogens from the host with a limited number of cell surface receptors. This challenge is compounded because microbial pathogens have the ability to mutate as a mechanism to escape host recognition. The innate immune system has met this challenge through recognition of evolutionarily conserved structures on pathogens that are not present in higher eukaryotes (PRRs). These molecular motifs (MAMPs) have essential roles in the pathogen’s ability to evade host defense and thus are not subject to high mutation rates. MAMPs are shared among various species of microbes but are not expressed by the host. Although many PRRs have been known for years, it was not clear how the innate immune system functioned until the discovery of TLRs, which have proved to be critical for recognition of microbes by the innate immune system and for bridging the innate and acquired immune responses. Interestingly, within the periodontal tissues, the expression of various TLRs appears to be increased in severe disease states.29 Table 25-1 illustrates PRRs and the host cells that express them and their ligands (MAMPs) present in microorganisms.
TABLE 25-1 Pattern-Recognition Receptors (PRRs), Host Cells (Mouse or Human) that Express Them, and Their Ligands (MAMPs) Present in Microorganisms Relevant to Periodontal Diseases
However, there are other PRRs that can be secreted into the plasma as humoral proteins and others that are localized in the cytoplasm as intracellular sensors. Soluble PRRs include various proteins, including collectins, ficolins, and acute-phase pentraxins (e.g., C-reactive protein) that represent functional ancestors of antibodies. These soluble mannose-binding receptors can interact with structures from microbes and activate the complement system via mannan-binding lectin-associated serine kinase (MASP) pathway.269 In fact, direct interaction between complement precursors and microbes can initiate activation through an unclear mechanism that culminates with opsonization and lysis of the microbes. Another example of soluble PRRs is the lectin-complement system, which is involved in recognizing the microbe by the carbohydrate-binding lectin, leading to its subsequent opsonization and phagocytosis.331
Nucleotide-oligomerization domain (NOD) protein-like receptors represent cytoplasmic PRRs and are characterized by C-terminal leucine-rich repeats (LRR domain, similar to the TLRs), an N-terminal caspase-activating recruitment domain (CARD), and a nucleotide-binding domain (NBD). These were initially described as cytosolic TLRs, analogous to the R proteins present in plants.70 NOD proteins are capable of recognizing different peptidoglycan molecules: Nod1 recognizes peptidoglycan containing meso-diaminopimelic acid (meso-DAP) fragments present in most gram-negative and some gram-positive bacteria,106 whereas Nod2 recognizes muramyl dipeptide (MDP), which is found in peptidoglycan from both gram-negative and gram-positive bacteria.207 More recent evidence indicates that NOD proteins, specifically Nod1 and Nod2, are involved in activation of inflammatory gene expression90 and even LPS recognition independently of TLR.152 The relevance of NOD proteins for the immune response is demonstrated by the association between genetic mutations on Nod1 and Nod2 and development of allergic conditions and Crohn’s disease, respectively.148,153 Another type of intracellular PRR is represented by a cytoplasmic protein family, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which play a critical role in recognizing viral ribonucleic acid (RNA) and induction of type I interferon (IFN) expression. However, since the role of retroviruses in periodontal disease is not clear, RLRs will not be discussed here (for a review of RLRs, see Yoneyama and Fujita340).
All of these different PRRs represent the necessary armamentaria for recognition of MAMPs by the host and are expressed by a variety of cells that play a role in innate immunity. Importantly, our understanding of the roles of PRRs in inflammation and immune responses has been expanded, so it is now appreciated that these receptors not only recognize various MAMPs to activate innate immune response, but they can also bind to endogenous molecules derived from damaged tissue and have a role in inflammation and adaptive immune responses. The cells involved in innate immunity include macrophages and polymorphonuclear cells as professional phagocytes with the primary function of engulfing and destroying microbes; dendritic cells as professional antigen-presenting cells and activators of adaptive immunity; and natural killer (NK) cells, the innate cytotoxic lymphocytes that recognize and kill host cells that are altered (e.g., tumor cells) or infected with viruses. However, other cell types can also play important roles in innate immunity, since they are able to recognize MAMPs through their PRRs and respond by expressing biologically-active molecules, such as cytokines and matrix metalloproteases (MMPs), that will have an effect on homeostasis of the host tissues in the periodontal microenvironment. Resident, “nonprofessional” cells such as fibroblasts and osteoblasts are also capable of producing a variety of cytokines, such as interleukin-6 (IL-6), prostaglandin E2 (PGE2), MMPs, and receptor activator of NF-κB ligand (RANKL). Table 25-2 presents these biologic mediators elicited by different MAMPs in resident and nonresident cells involved in the pathogenesis of destructive periodontal disease. Because of the sheer proportion of fibroblasts in the periodontal tissues and also to the proximity and relevance of both fibroblasts and osteoblasts to nonmineralized and mineralized tissue turnover, respectively, these cells can play important roles in innate immunity during periodontal diseases. Epithelial cells represent the initial point of contact with MAMPs in the periodontium and play an important role in innate immunity not only because of their function as a physical barrier, but also because these cells are equipped with PRRs and respond to MAMPs by secreting various cytokines and chemokines, including IL-8 and antimicrobial peptides (e.g., beta defensins, cathelicidins).69,156
TABLE 25-2 Biological Mediators Elicited by Different MAMPs in Resident and Nonresident Cells Involved in the Pathogenesis of Destructive Periodontal Disease
Despite the alleged nonspecificity of the innate immune response, it has long been known that there are important quantitative and qualitative differences in cytokine release on stimulation with gram-positive or gram-negative bacteria.
According to the current paradigm of the microbial etiology of periodontal disease, disease initiation depends on shifts in the microbial population of the dental biofilm toward a more complex flora that includes gram-negative and anaerobic species. This implies that some bacterial species in the dental biofilm may not play a role in periodontal disease and in fact, clinical periodontal health is frequently observed in spite of the presence of a dental biofilm. However, the fact that different species of bacteria share common MAMPs (e.g., CpG deoxyribonucleic acid [DNA], LPS, and peptidoglycans) that can potentially trigger an innate immune response suggests that tolerance mechanisms may counterbalance the vigilance aspect of innate immune responses to allow the presence of these commensal, nonpathogenic bacteria. Tolerance mechanisms have been extensively studied in the gut epithelium, since the intestinal mucosa is continuously exposed to innocuous environmental antigens and commensal microorganisms that live in symbiosis with the host (for review, see Artis14). The critical importance of these tolerance mechanisms is exemplified by the fact that its dysregulation is associated with the pathogenesis of various inflammatory conditions, including inflammatory bowel disease and intestinal cancer.40,165
There are some important differences between the oral epithelium and the intestinal mucosa, especially the presence of specialized immune cells subjacent to the mucosal epithelium in the intestines (gut-associated lymphoid tissues [GALT] and Peyer’s patches); however, the concept of a tolerance mechanism that renders the immune system hyporesponsive to commensal bacteria, while retaining the capacity to respond to pathogenic organisms has also been investigated in oral epithelial cells. A comparison of the responses of oral epithelial cells to nonperiodontopathic and periodontopathogenic bacteria demonstrated that, in general, commensal bacteria (such as Streptococcus gordonii or Streptococcus sanguinis) induce expression of antimicrobial peptides without inducing the chemoattractant cytokine IL-8, whereas periodontopathogenic bacteria from the “orange complex” (Fusobacterium nucleatum and Prevotella intermedia) induce strong expression of both antimicrobial peptides and IL-8. Interestingly, the bacteria that are more strongly associated with periodontitis (“red complex” organisms Treponema denticola, Tannerella forsythia, and Porphyromonas gingivalis) tended to suppress the innate immune response by inhibiting the expression of antimicrobial peptides, IL-8, or both simultaneously.156,179,324 Tolerance mechanisms may involve the activation of different signalling pathways, since the activation of NF-κB (which is associated with the expression of many inflammatory genes) was not required for the expression of antimicrobial peptides by oral epithelial cells after stimulation with commensal bacteria, as opposed to the periodontopathogenic microorganisms.56 There is still much to be explored in these tolerance mechanisms, but it is conceivable that commensal bacteria may be beneficial to the host as they induce expression of antimicrobial peptides, which will clear some of the nonpathogenic and pathogenic bacteria while allowing nonpathogenic periodontal bacteria that are resistant to antimicrobial peptides, such as S. gordonii and S. mutans,156,251 to establish themselves in the oral biofilm. The weak induction of IL-8 may function to maintain immune vigilance without causing too much collateral damage to the host tissues. On the other hand, pathogenic bacteria (such as the orange complex microorganisms, F. nucleatum and P. intermedia) that induce both antimicrobial peptides and IL-8 expression may be limiting their survival in the biofilm. Interestingly, the inhibition of expression of antimicrobial peptides and IL-8 in epithelial cells by red complex bacteria (such as P. gingivalis and T. denticola) can represent host-evading strategies that allow them to survive and establish themselves in the oral biofilm.44
Activation of the innate immune system is critical for lymphocyte activation and other immune cells to help clear the infectious microorganisms. However, exuberant production of proinflammatory cytokines leads to severe pathology, including alveolar bone loss.17,76,77,113,114 To help prevent deleterious effects of TLR activation, a number of signalling mechanisms have evolved. These mechanisms include downregulation of surface TLR receptor expression, transcriptional induction of negative regulators such as IL-1 receptor-associated kinase (IRAK-M), suppressor of cytokine signalling-1 (SOCS1), and SH2-containing inositol phosphatase (SHIP), and production of antiinflammatory cytokines, including IL-10 and transforming growth factor-beta (TGF-β).191 In contrast to the release of proinflammatory cytokines and mediators, production of these negative immune regulators occurs over a much longer time frame, providing a vital role in controlling the extent of proinflammatory and antiinflammatory mediators in a proper temporal sequence.191
Cell Signalling Pathways and the Expression of Biologically-Active Mediators in the Innate Immune Response
Once MAMPs are recognized by the appropriate PRRs, a signal is initiated within the cell. This signal is transduced through the cytoplasm and nucleus more commonly by sequential posttranslational modification (e.g., phosphorylation) of a cascade of kinases that are constitutively expressed and ultimately will determine the cell response to the MAMPs detected (Figure 25-2).
TLRs are single-pass transmembrane proteins with N-terminal presenting LRRs that are responsible for the recognition of their ligands and with a C-terminal cytoplasmic domain that is very similar to the cytoplasmic region of the IL-1 receptor,3 which is called the Toll/IL-1 receptor domain (TIR). Thus, subsequent to the recognition of a ligand by TLRs, the signal generated uses pathways similar to those utilized by the IL-1 receptor. TLR signalling was originally described in the context of the activation of the IFN regulatory factor IRF family of transcription factors and NF-κB, leading to the expression of IFN-γ and early-response inflammatory genes, respectively.
Interestingly, the participation of at least four adaptor proteins (MyD88, TRIF, Mal/TIRAP, and TRAM) containing TIR domains that can be recruited by activated TLRs results in important branching of the signal transduction and yields a significant flexibility to TLR signalling by allowing cross-talk with other pathways, notably the MAP kinase pathway. These adaptor proteins are recruited to TLRs by homophilic interactions between their TIR domains and are utilized differently by the TLRs. TLR5, TLR7, and TLR9 were shown to depend on recruitment of MyD88 to signal,136,142 whereas TLR3 is the only TLR that does not use MyD88.337 TLR4, on the other hand, can use all four adaptor proteins: MyD88, TRIF, Mal/TIRAP, and TRAM.336-338 Even though activation of the canonical NF-κB pathway is usually affected by all TLRs, the timing of NF-κB activation,166,167 as well as the additional signalling pathways that are activated by the branching of the signal, varies among TLR receptors and with the participation of different adaptor proteins (Figure 25-2). These variations will ultimately affect the biologic result in terms of gene expression as demonstrated by the finding that even though NF-κB activation is observed after TLR4 stimulation by LPS, this may or may not result in inflammatory gene expression, depending on the adaptor protein used. In wild-type cells, LPS stimulation results in inflammatory cytokine expression, whereas in MyD88-deficient cells, LPS fails to induce cytokine expression. In the absence of MyD88, activation of NF-κB occurs with delayed kinetics in comparison to wild-type cells.166
Figure 25-2 Pattern recognition receptors (PRRs) and innate immune signalling. TLR2, TLR4, and TLR9 are depicted as examples of TLR receptors expressed in cells of the periodontal tissues. On ligand binding, all TLRs (except TLR3) recruit adaptor protein MyD88 and activate common upstream activator (IRAK/TRAF6 and TAK1) of NF-κB and MAPKs. TLR4 may also activate NF-κB independently of MyD88 and with delayed kinetics (red dotted line). Nod1/Nod2 are cytosolic PRRs that recognize peptidoglycan fragments of bacterial cell walls and may amplify the TLR-induced activation of signalling pathways. Activated NF-κB and MAPKs translocate to the nucleus and bind to their motifs (NF-κB, AP-1, respectively) in the promoter region of target genes (including early-response and inflammatory genes) and induce their transcription into mRNA, which will ultimately lead to increased cytokine production. p38 MAPK is also involved with post-transcriptional regulation of proinflammatory genes (e.g., IL-6, Cox-2) by modulation of mRNA stability in the cytoplasm. TLR, Toll-like receptor; CD14, cluster of differentiation 14 molecule; MD2, myeloid differentiation protein 2; MyD88, myeloid differentiation primary response gene 88; IRAK, interleukin-1 receptor-associated kinase; TRAF6, TNF receptor-associated factor 6; TAK1, TGF-beta activated kinase 1; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; AP-1, activator protein-1).
The shift in the microbial population present in the oral biofilm from predominantly gram-positive to gram-negative bacteria that is associated with the onset of periodontal disease may lead to different patterns of immune response as a result of the type of TLR predominantly activated. Gram-positive bacteria were shown to activate TLR2, which induced increased expression of IL-8, whereas gram-negative bacteria activated predominantly TLR4, resulting in increased expression of tumor necrosis factor alpha (TNF-α).312
However, some gram-negative microorganisms present in the oral biofilm and associated with periodontal disease are rather unique in their capacity to activate NF-κB via preferential utilization of TLR2.45 Recently, it was reported that most gram-negative bacteria associated with periodontal disease, including P. gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescences, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Veillonella parvula are all capable of activating TLR2, whereas the latter two microorganisms also activate TLR4.172 Even though all these disease-associated microorganisms activate TLR2 signalling, this pathway can also be activated in vitro by microorganisms present in an oral biofilm composed primarily of gram-positive bacteria and which are common colonizers of the oral biofilm and not associated with clinical signs of periodontal disease.343 The fact that TLR2 is activated by both pathogenic and nonpathogenic microorganisms is an interesting finding and suggests differences in the utilization of adaptor proteins and/or concomitant activation of other TLRs by different MAMPs expressed by the various bacterial species that are present in an oral biofilm associated with disease. These differences can lead to the activation of different signalling pathways and subsequent modulation of the host response.
NOD-like receptors include 23 genes in humans, and even though some of these genes are primarily expressed in macrophages and polymorphonuclear leukocytes (PMNs), the best studied and characterized members Nod1 and Nod2 are expressed by a wide variety of cells, including epithelial cells,318,319 monocytes/macrophages,230 and dendritic cells306 (see Table 25-1). The exact mechanism by which these proteins recognize their ligands is still unknown, and there is no evidence for direct interaction between the ligands and NOD proteins. According to the proposed mechanism of activation of NOD proteins, they are kept in an inactive state by intramolecular interactions between the C-terminal LRR domain and N-terminal fragment CARD and NOD domains. Ligand recognition results in conformational changes that relieve the autoinhibitory intramolecular interactions and allow NOD–domain-dependent nucleotide binding and oligomerization.145 Stimulation of Nod1 and Nod2 will result in activation of NF-κB and mitogen-activated protein kinases (MAPKs), similarly to the activation of TLRs (see Figure 25-2); however, the signal transduction pathways require different signalling intermediates. Activation of Nod2 leads to recruitment of a kinase called receptor-interacting protein 2 (Rip2, also known as RICK), which will bind directly to inhibitor of NF-κB kinase gamma (IKKκ, also known as NF-κB essential modulator [NEMO]) and promote its ubiquitination and activation of the catalytic regulatory subunits of the inhibitor of NF-κB kinase (IKK) complex. Once activated, IKK phosphorylates the inhibitor IkB leading to its degradation by the proteasome, releasing NF-κB and allowing its translocation to the nucleus where it will affect expression of various inflammatory genes.134,234 In this pathway, Rip2 has been shown to be required for Nod1/Nod2 activation of NF-κB,234 but interestingly its kinase activity is not essential.131 The same signalling intermediate Rip2 has also been shown to be involved in Nod1/Nod2-induced MAPK activation by mediating the recruitment of upstream activator of MAPK, TGF-β-activated kinase 1 (TAK1).131 However, even though Rip2 and TAK1 are required for Nod1/Nod2-mediated activation of MAPK, the intermediate steps in the activation of this pathway are not well known.234,281
Since TLRs and NOD proteins are PRRs involved in recognition of bacteria and considering that the signals generated by their activation converge to the same signalling pathways, there may be a synergistic or “amplifying” effect for simultaneous NOD and TLR activation by MAMPs. This cooperation between different PRRs in the activation of NF-κB and MAPK-signalling pathways may increase the sensitivity and potency of the host response to the presence of bacteria.90,91,323 Indeed, there is evidence demonstrating that activation of Nod1/Nod2 has synergistic effects with TLR signalling on the production of proinflammatory cytokines (including IL-1, IL-4, IL-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor [GM-CSF], and TNF).91,234,306,323 Moreover, it has been suggested that Nod1/Nod2 signalling augments t-helper 1(Th1) polarization induced by TLR signalling, except for TLR2 signals, which inhibit Th1-type cytokines because of IL-10 production.306
Besides their role as PRRs and synergism with TLR signalling, other members of the NOD family of proteins can also affect innate immune response by their role in the formation of the inflammasome, which is an intracellular multiprotein complex that activates caspase-1. Activated caspase-1 will process the inactive pro–IL-1β and pro–IL-18 forms produced and convert them into the biologically active forms secreted.184
Adaptive Immunity in Periodontal Diseases
Activation of Adaptive Immunity in Periodontal Diseases
Besides its role in the immediate detection of infectious agents and toxic/degradation (“danger”) products to elicit an inflammatory response and activate effector processes aimed at the elimination or confinement of microbial invaders, innate immunity also has the pivotal role of initiating and modulating adaptive immune responses. Thus adaptive immunity requires the innate “branch” of immunity to be activated; however, in vivo, this didactic distinction does not exist and the immune response should be understood as a continuum, a constantly adjusting host reaction to the presence of microbes and their MAMPs. In the context of this chapter, the signalling and molecular aspects of the host-microbe interactions related to the adaptive immune response will be emphasized.
Innate immune mechanisms and signalling are not “turned off” once the adaptive immune response is activated but rather are “supplemented” by the latter in the defense against microbes. On the other hand, cells from adaptive immune response also express PRRs and respond to MAMPs, which further emphasizes the dynamic interaction between the two “arms” of the immune response.
In T lymphocytes, TLR agonists have been shown to modulate the expression of costimulatory receptors (such as CD28, CD69, and CD152) and increase their proliferation and IFN-γ production, suggesting that microbes and their MAMPs may also have a direct functional role in the regulation of adaptive immunity.47,292 This is especially important to realize in the context of periodontal disease, which is a chronic inflammatory condition and as such has a dense lymphocytic infiltrate. Ever since the classic studies of the host response in periodontal disease, including the recognition of immunoglobulin-producing plasma cells in the gingival tissues of patients with periodontal disease,43 the classic descriptions of initial, established, and advanced lesions233 and the characterization of the inflammatory infiltrate in the dog model,192 the nature of the inflammatory infiltrate and its association with the inflammatory response and ultimately the quiescence or progression of disease, has been debated.
The adaptive immune response is characterized by the activities of pathogen-specific B and T lymphocytes; however, the cell type primarily responsible for translating innate signals into adaptive immunity is the dendritic cell (DC). In fact, adaptive immunity initiates with DCs recognizing MAMPs in the sites of infection and subsequently migrating into the regional draining lymph nodes to present the processed antigen peptides in the context of major histocompatibility complex (MHC) molecules to naive T lymphocytes. Once inside the lymph nodes, DCs migrate to the T cell areas, seek out antigen-specific T cells, and induce their activation and differentiation into effector cells (helper or cytotoxic cells). The maturation process induced by MAMPs that leads to the migration of DCs to the lymph nodes involves modulation of the expression of various chemokine receptors that render DCs less responsive to inflammatory signals and more responsive to lymphocyte-derived signals. Notably, LPS-induced maturation of DCs has been shown to result in the downregulation of “inflammatory” CCR1 and CCR5 (which responds to macrophage inflammatory protein-1α [MIP-1α], for example) and upregulation of CXCR4 and CCR7 (which respond to CXCL12 and CCL19, respectively).274 These changes ultimately control DC trafficking and migration to the lymph nodes and represent one mechanism, whereby modulation of cell signalling pathways (MAMPs activating PRR signalling versus inflammatory cytokine signalling) can influence the host response. The profound phenotypic changes that DC cells undergo in the process of activation by MAMPs and conversion into antigen-presenting cells is a multistep process that includes not only trafficking and migration into lymph nodes, but also the expression of high levels of MHC bearing the processed microbial-derived peptides that will engage T cell receptors in naive T cells, the expression of costimulatory membrane-bound molecules (such as CD80 and CD86), which are important for T-cell survival and proliferation, and finally the production of mediators (such as IL-12) that act on T cells and induce their terminal differentiation into an effector cell type and their profile of cytokine expression. Ultimately, the integration of these signals (MHC-antigen; costimulatory molecules, cytokines) will determine the fate and nature of the adaptive immune response.161
Interestingly, many of the features of DC activation can be induced by inflammatory cytokines in the absence of MAMPs and PRR signalling. This fact is interpreted by some as supportive of the “danger model” of immunity, in which endogenous danger signals (e.g., toxic products from necrotic, apoptotic, or infected cells, inflammatory cytokines, etc) can also trigger an immune response, similarly to exogenous MAMPs.30,204 There are two important arguments against this concept, as follows:
The signalling aspects involved in DC maturation and activation are especially interesting since most PRR and inflammatory receptor signals converge to similar pathways (NF-κB and MAPK). In spite of similar signalling pathways activated by MAMPs and inflammatory cytokines, MyD88-defficient dendritic cells stimulated with inflammatory cytokines fail to produce IL-12 and other inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-β),143 which will have important consequences for the differentiation and activation of naive CD4+ and CD8+ T cells67 and also indirectly affect activation of B cells and the humoral response.297 Among the possible reasons for this differential role of MAMPs and inflammatory cytokines as external signals in spite of the activation of similar signalling pathways are the kinetics of the activation of the signalling pathways and also the other signalling pathways that are simultaneously activated by each external signal. Integration of multiple signal pathways may be required for a given profile of cytokine expression by DCs. Specifically in periodontal diseases, both MAMPs and inflammatory cytokines are usually present to fully activate the DCs, which suggests that there is no impairment to a competent activation of adaptive immunity.
Host-Microbe Interactions in Adaptive Immunity
Since humans are exposed to bacteria in various mucosal sites, including oral mucosa, shortly after birth and this exposure continues throughout our lives, it is reasonable to expect a chronic presence of adaptive immune cells as part of a vigilance mechanism in these sites chronically exposed to microbes and their MAMPs. The crucial role of adaptive immunity to periodontal disease progression is currently undisputed; however, the mechanisms are not fully understood. According to the current paradigm, DCs activated by MAMPs and inflammatory cytokines (also induced by MAMPs in innate immune/resident cells) will initiate an adaptive immune response by driving naive T lymphocytes into a CD8+ (for cytotoxic response) or CD4+ with Th1 or Th2 phenotypes. Recently, more pieces have been added to the puzzle, including the regulatory T lymphocytes (Tregs), which appear to have their inhibitory functions suppressed by activated DCs. Activated T cells and their “specific” cytokine profiles will modulate the inflammatory response and also the activation of B lymphocytes.
The helper subtype of T cells appears to be particularly relevant for periodontal diseases, as shown by studies in which mice lacking CD4+ cells had decreased severity of bone loss induced by P. gingivalis challenge, whereas depletion of CD8+ cells did not have the same effect.24 There has been a long debate on the cytokine profile associated with periodontal disease, and it has been suggested that the outcome (progression of disease or quiescent state) is determined by the predominant T lymphocyte cytokine profile (Th1 or Th2) in the inflamed tissues.144 However, the cytokine profile associated with periodontal disease in vivo varies and includes both Th1- and Th2-type responses. Some studies using in vivo models have associated a Th1-type cytokine profile with progressive periodontal diseases.276,288,311 Compelling evidence for the role of Th1-type response in periodontal disease progression comes from studies in which in periodontal bone loss in rats challenged with A. actinomycetemcomitans was associated with the introduction of T lymphocytes of the Th1 subtype but not with the Th2 subtype T lymphocytes.125 In fact, introduction of T lymphocytes of the Th2 subtype in T cell-depleted rats challenged with P. gingivalis resulted in decreased bone loss, suggesting a protective role for Th2-type responses in periodontal disease.84 Others reported that even if the Th2 response is not protective of bone loss, it does not aggravate the bone loss induced by P. gingivalis challenge, as observed with the Th1-type response.187 In contrast, a Th2 cytokine profile has also been reported as associated with progressive periodontal disease by both in vitro11,290 and in vivo314,339 studies.
Recently, both B and T lymphocytes were shown to express PRRs, more specifically certain types of TLRs. There is still some controversy over the type of TLR and the level of expression, which can be attributed to various experimental methods used, including the procedure to purify lymphocytes, the model (murine or human), and the analysis method (flow cytometry, real time polymerase chain reaction [PCR]). Nevertheless, messenger RNA (mRNA) for TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 was detected in human peripheral T lymphocytes, although the expression levels are highly variable among different studies.47,81,141,345 In spite of the controversy, there are some studies demonstrating a functional role for these PRRs expressed in lymphocytes. In naive T cells, TLR2 and TLR4 may not be functional, since their expression was demonstrated intracellularly,19,177,197 whereas TLR5 was detected in the cell surface.64 However, expression of cell-surface (and functional) TLR2 and TLR4 is regulated in naive human T CD4+ lymphocytes by T-cell receptor (TCR) activation, and the same is true for TLR2 in murine CD4+ and CD8+ T cells.177,193 In contrast, TLR5, constitutively expressed on the cell surface of human CD4+ T lymphocytes, is downregulated by TCR stimulation.64 The mechanism for the TCR-mediated modulation of TLR expression is not known; it may involve induced translocation of intracellular TLRs to the cell surface, or de novo synthesis of membrane-bound TLRs.164 Current evidence suggests that T cells can respond directly to MAMPs through TLR and that TLR signalling in T cells has a costimulatory effect with TCR activation. TLR2 signalling costimulates proliferation and cytokine production by CD4+177,193 and murine CD8+ T cells.62 TLR3 signalling increased survival of activated murine CD4+ T cells,103 whereas TLR9 signalling increased not only survival but also proliferation of CD4+ T cells.104 TLR4 stimulation increased perforin expression in CD4+ T cells, suggesting a role for LPS in the induction of a cytotoxic response.250
Expression of TLRs may also vary in different T-cell subsets. Tregs (CD25+/CD4+) express higher levels of TLR564 and as opposed to CD4+/CD25− T lymphocytes, also express TLR8.243 Treg cells have an important role as negative regulators of immune response and consequently in immune tolerance mechanisms. Their function requires precise control to maintain adequate and effective T cell-mediated immunity.272 Even though the mechanisms for the suppressive effect of Tregs are not well known, they require expression of transcriptional repressor FoxP3.268 During the activation of adaptive immunity, activated DCs not only polarize naive CD4+ T lymphocytes to a Th1 or Th2 subtype, but they also inhibit Tregs. There is evidence that this inhibition of Treg suppressive function involves production of IL-6 by activated DCs and other innate immune cells.225,236 Interestingly, direct stimulation of TLR2 or TLR8, together with TCR activation in Tregs results in increased proliferation and inhibition of FoxP3 expression and consequently of their immune suppressive activity.193,243,305 There is also evidence of modulation of Treg activity according to dose-dependent stimulation of TLR signalling. Low concentrations of flagellin (TLR5 ligand) enhanced the suppressive activity of Tregs (as in an immune tolerance mechanism), whereas higher concentrations of flagellin increased proliferation and cytokine production in CD4+ T cells.64 TLR4 signalling also appears to enhance Treg suppressive functions through IL-10 and TGF-β dependent mechanisms.189
The proportion of B cells is reported to be larger than that of all subsets of T lymphocytes.32 In fact, B–cell-derived plasmocytes and B lymphocytes represent the absolute majority of infiltrating cells in diseased periodontal tissues, and their relative proportion appears to be higher in sites with severe periodontal disease in comparison to sites with moderate-to-mild periodontitis. Besides their classic role as producers of immunoglobulins that identify and bind to MAMPs, B cells also participate as antigen-presenting cells and modulators of host response through the production of cytokines such as TNF-α, IL-6, IL-10, TGF-β, and RANKL.33 In fact, the relevance of B lymphocytes for the pathogenesis of periodontal disease and in particular, for alveolar bone loss has been demonstrated in murine models.124,127 The role of the humoral response in periodontal disease has not been clear until now, and the importance of B cells for periodontal disease pathogenesis is probably largely the result of cytokine production.55,168 Antibodies are considered as primarily defense mechanisms, aimed at neutralizing directly (through inactivation of antigens with or without complement activation) or indirectly (through opsonization of antigens and facilitating phagocytosis) the microbes. However, there is no definite explanation as to why the elevated levels of immunoglobulins against periodontopathogenic bacteria are not sufficient to ameliorate periodontal disease or to prevent its recurrence. Elevated serum titers of antibodies against P. gingivalis have been detected before but did not prevent alveolar bone loss in a murine model of periodontal disease.23 Indeed, elevated levels of immunoglobulins may actually aggravate local destruction of periodontal tissues through complement activation or through self-reactive antibodies.32 It has also been suggested that the high immunoglobulin G (IgG) serum titers against periodontal bacteria may be implicated as one of the possible biologic links between periodontal diseases and systemic diseases such as cardiovascular disease227 or rheumatoid arthritis.262 B cells have been shown to express various TLRs, including TLR1, TLR2, TLR4, TLR6, TLR7, TLR9, and TLR10.141 Recent evidence indicates that a significantly higher proportion of B cells from periodontal tissues and blood from periodontal disease patients expressed TLR4 in comparison to those of periodontally healthy individuals.289 This suggests that expression of some TLRs may be induced, likely by the inflammatory environment resulting from the initial activation of innate immunity by MAMPs. Thus cytokines produced by innate immune response and also the presence of MAMPs would induce TLR expression in B cells, which in turn renders these cells more responsive to the ubiquitously present MAMPs in the periodontally diseased tissue. This concept is supported by the fact that resting B cells express primarily TLR1 and TLR9, with lower proportion of resting B cells expressing TLR2, TLR3, and TLR4.73 Activation of B cells in vitro by CD40 stimulation also increased expression of TLR7, TLR9, and TLR10, and interestingly, costimulation with CD40 ligand and CpG bacterial DNA had a synergistic effect on this induction.41
Importantly, TLR expression by B lymphocytes indicates that similarly to the T lymphocytes, these cells can respond to MAMPs and play a role in the modulation of both innate and adaptive arms of immune response, further stressing the continuity and cooperation between these two aspects of the host response. This cooperation is demonstrated by the reduced antibody production in humans and mice with reduced expression of TLR1 or TLR27; increased proliferation; activation of NF-κB, p38, and c-Jun N-terminal-activated kinases JNK MAPK; and also increased expression of costimulatory molecules (CD86, CD40, or MHC-II) and IL6 by B cells stimulated with TLR9 agonist.129
Pathobiology of Periodontal Disease Progression
Host response to periodontal infection requires expression of a number of bioactive agents, including proinflammatory and antiinflammatory cytokines, growth factors, and enzymes that are the result of the activation of multiple signalling pathways. This activation of intracellular signalling may initiate exclusively as an innate immune response associated with PRR-mediated sensing of MAMPs. However, the biologic mediators expressed as a result of PRR-signalling include costimulatory molecules involved in the induction of adaptive immunity.36 This results in a cascade of events that will establish very complex cytokine and signalling networks.
Innate immunity and inflammation are not synonymous; however, inflammation arises primarily in response to infection. A better understanding of direct host-microbe interactions is especially important to understand how inflammation is initiated in response to microorganisms in the dental biofilm. In this sense, PRR signalling is considered the most important interface between the host and the microbes,36 and as discussed previously in this chapter, it can modulate both innate and adaptive aspects of the host response.
Cytokines and Mediators of Inflammation
Inflammation involves a number of biochemical and cellular alterations. An inappropriate inflammatory response is the cause of many common diseases, including periodontitis. The local inflammatory reaction, in response to bacteria in the dental biofilm, is characterized by an initial increase in blood flow, enhanced vascular permeability, and the influx of cells from the peripheral blood to the gingival crevice. These initial events are triggered by bioactive molecules (such as histamine, bradykinin, PGE2, and nitric oxide) and produced by innate immune cells and resident, nonimmune cells present in the periodontal tissues. PMNs or neutrophils attracted to the area by other bioactive molecules (e.g., IL-8) migrate through the epithelial lining of the gingival sulcus to be the initial defense against invading plaque bacteria and their by-products.195 These cells are nonspecific phagocytes responsible for an acute and rapid defense. Subsequently, there is an increase in the number of monocytes/macrophages, as well as an influx of T and B cells to the area.97
Once activated by cytokines, bioactive molecules, and MAMPs present in the area, infiltrating cells produce other inflammatory mediators that modulate the activity of other cells and affect homeostasis of nonmineralized and mineralized tissues in the periodontium59,111 (Table 25-2 and Figure 25-3). Cytokines responsible for early responses to microbial aggression include IL-1α, IL-1β, IL-6, and TNF-α.185 Other proinflammatory mediators include leukemia-inhibiting factor (LIF); IFN-γ; oncostatin M (OSM); ciliary neurotrophic factor (CNTF); TGF-β; GM-CSF; IL-11, IL-12, IL-17, IL-18, IL-8, and a variety of other chemokines that attract inflammatory cells.49,226,275,276 The net effect of an inflammatory response is determined by the balance between proinflammatory and antiinflammatory cytokines such as IL-4, IL-10, IL-13, IL-16, IFN-α, TGF-β, IL-1Ra, GM-CSF, and soluble receptors for TNF or IL-6.112,186
Figure 25-3 Stimuli and factors regulating osteoclastogenesis. Cytokines (interleukin-1 [IL-1], tumor necrosis factor alpha [TNF-α], macrophage-colony stimulating factor [M-CSF]) produced by resident cells (bone stromal cells, fibroblasts, osteoblasts) and immune cells (monocytes, T- and B-lymphocytes) are key regulators of this process. Osteoprotegerin (OPG) acts as a decoy receptor that prevents receptor activator of nuclear factor (NF)-κB ligand (RANKL) from binding to its receptor activator of NF-κB (RANK) on precursor cells and downregulate osteoclastogenesis. Bacterial lipopolysaccharide (LPS) and other microbial-associated molecular patterns (MAMPs) may directly or indirectly initiate expression of RANKL and osteoclastogenesis.
Patients with periodontal inflammation have high concentrations of TNF-α, IL-1β, RANKL, and MMP-13* in the gingival crevicular fluid (GCF). Increased levels of IL-1β, IL-2, IL-6, IL-17, TNF-α, and IFN-γ in gingival tissues are also associated with destructive periodontal disease.110,188
However, in spite of an abundance of publications investigating cytokine profiles associated with periodontal disease, the definitive description of the cytokine response still remains elusive. Among the reasons for the variability of the reports on the cytokine profile are the difficulties in defining periodontal disease and especially in the identification of actively progressing disease sites (i.e., those with ongoing attachment loss and alveolar bone resorption). A gene-expression profiling (or transcriptome analysis) of changes in mRNA levels of over 38,000 genes with 54,000 DNA probes was performed in healthy and diseased periodontal tissues, and demonstrated that over 12,000 probes were differentially regulated in periodontal diseased tissues, including genes involved in inflammation, apoptosis and angiogenesis.78 These data agree with the evidence demonstrating that the cytokine profile associated with periodontal disease in vivo varies and includes both Th1- and Th2-type cytokines. IL-1α, IL-1β, IL-8, and TNF-α mRNA were detected in macrophages present in inflamed gingival tissues,201 whereas Th2 cytokine IL-4 and pleiotropic IL-6 protein were also observed in diseased periodontal tissues.339 A characteristic cytokine profile has been associated with each type of periodontal disease (i.e., inflammation of marginal soft tissues without active bone resorption [gingivitis] or with active bone resorption [periodontitis]). Thus expression of Th1-type cytokines has been associated with gingivitis, whereas Th2 cytokines were found in higher levels in periodontitis-affected tissues,186,314 even though this distinction was not clear because both Th1 and Th2 cytokines were produced in gingivitis- and periodontitis-affected tissues; and the predominant profile may actually represent the current activity of tissue destruction.101,286,335 The relevance of cytokines as biologic mediators of periodontal disease progression is demonstrated by studies showing that conventional periodontal therapy aimed at the reduction of the bacteria and associated metabolic by-products result in their decrease, as shown for IL-1β4,61,313 and TNF-α.154
Once the immune and inflammatory processes are initiated and the complex cytokine network is established, various inflammatory molecules that play a direct role in the degradation of both nonmineralized and mineralized tissues of the periodontium are produced in response to MAMPs and/or to the host-derived cytokines.59,111 One such family of inflammatory molecules is the MMPs, which are released from different cell types present in the lesion, including macrophages, leukocytes, and fibroblasts or other resident cells.178 MMPs are a family of neutral proteases that are zinc/calcium-dependent with an essential role in extracellular matrix (ECM) turnover and degradation. Their activity is regulated at multiple levels (transcriptional, posttranscriptional, and posttranslational) and also by endogenous inducible inhibitors, the tissue inhibitors of MMPs (TIMPs). Collectively, MMPs are capable of degrading virtually all components of ECM, and increased levels have been associated with periodontal disease in both humans61,87,89,180 and animal models.74,102 The same difficulties associated with the determination of disease activity apply to the determination of MMPs in periodontal disease, even though the trend seems to have a positive correlation (i.e., MMPs increase with inflammation and disease activity108) and their detection in saliva has been recently suggested as one possible host-response biomarker of periodontal disease.254 Interestingly, the role of individual MMPs in periodontal disease progression may differ, as MMP-8 was recently shown to have a protective effect on alveolar bone loss induced by P. gingivalis infection in mice182; whereas MMP-13 expression was positively correlated with the severity of inflammation in ligature- and LPS-induced experimental periodontitis in rats74 and also with severity of periodontal disease in humans.291
The relevance of MMPs to the pathogenesis of periodontal diseases is supported by the decrease in bone loss associated with their nonselective inhibition in animal models of periodontal disease252,253 and especially by the improved clinical results observed after periodontal treatment associated with systemic inhibition of MMPs by a sub-antimicrobial dose of doxycycline.99,107,247,248
As periodontal disease progresses, the collagen fibers and connective tissue attachment to the tooth are destroyed, the junctional epithelial cells proliferate apically along the root surface, and these structural changes are reflected clinically as attachment loss and/or increased probing pocket depth. These changes represent not only an increased severity of inflammation but also an apical displacement of the inflammatory infiltrate, which thus is in close proximity to the alveolar bone. This affects homeostasis of bone tissue by triggering the resorptive process that represents the main characteristic of destructive periodontal disease. RANKL plays a pivotal role in bone resorption, since it is involved in osteoclast differentiation, activation, and survival.42,171,310 Osteoprotegerin (OPG) is the endogenous inhibitor of RANKL and functions as its decoy receptor, preventing binding of RANKL to the specific membrane-bound receptor expressed in osteoclast precursor cells (RANK).42,139,301 RANKL is produced as either a membrane-bound or secreted protein by fibroblasts, osteoblasts, chondrocytes, mesenchymal cells, and T- and B-lymphocytes, whereas OPG is secreted primarily by osteoblastic cells, bone marrow stromal cells, and fibroblasts (see Figure 25-3). The ratio between RANKL and OPG is the current paradigm for modulation of coupled bone turnover, and specifically in periodontal disease, this concept is supported by observations demonstrating that patients with advanced periodontitis present with higher levels of RANKL and lower levels of OPG than periodontally healthy patients.65,213 Further support to the relevance of RANKL/OPG cytokine system for bone homeostasis comes from studies in which local delivery of OPG decreases alveolar bone resorption in experimental periodontal disease models.54,159 However, other biologically-active mediators present in the complex network of cytokines established in the periodontal disease microenvironment can play important roles in periodontal disease pathogenesis and alveolar bone resorption, as indicated by studies targeting inhibition of TNF-α.57,79
It is important to consider that there is significant heterogeneity of clinical presentation of periodontal disease in terms of its extent, severity, progression, and response to treatment. This heterogeneity is thought to reflect the high variability of the levels of inflammatory cytokines and bioactive molecules produced by different individuals, which cannot be completely explained by differences in the microbial population of the dental biofilm. Based on susceptibility analysis, individual differences in the host response to MAMPs and to host-derived cytokines that are the result of genetic variations may play an important role in modulating the pathogenesis of periodontal diseases.341 This is supported by epidemiologic studies of the association between various genetic syndromes and periodontal diseases222 and the familial trend for occurrence of some types of aggressive periodontal disease.9,216 There are numerous studies correlating multiple variations in genetic information encoding genes and receptors involved in the immune response and inflammation with periodontal disease,93,151 and these genetic variations are also heterogeneic according to race and ethnic aspects of human populations.13,199,217,273,328 It is interesting to note that there is also evidence suggesting that genetic polymorphisms for the “effector” genes of MMPs18,52,246 and RANKL/OPG235,295 are associated with the severity and extent of periodontal disease. However, the strength of this association, especially for MMPs, is still controversial and may depend on specific types of MMPs, as other studies failed to demonstrate an association between polymorphisms for some MMPs and periodontal disease status.75,115,140
Another important aspect to consider is how the external environment affects the production of inflammatory mediators by the host. Thus, even though there is a “personalized genetic program” controlling how each individual responds to the microbial challenge, this is further modulated by transitory aspects of each individual’s life that can affect production of cytokines and inflammatory mediators, including hormonal imbalances, stress, acquired diseases, medications, and smoking. These are all factors that come into play in determining the individual’s response to the microbial challenge and ultimately, periodontal tissue destruction. Moreover, it has been recently suggested that genetic variations modulating host responses may represent a common trait for susceptibility to periodontitis and systemic diseases such as cardiovascular disease.283
Cell Signalling Events Modulating Inflammatory Mediator Expression
In the complex scenario of host-microbe interactions associated with periodontal disease, the signalling pathways originally shown to be relevant for recognition of stress, inflammatory, and infectious extracellular stimuli are of special interest. Production of cytokines and inflammatory mediators is usually a tightly-controlled process that is initiated by external stimuli or “signals” that are rapidly transduced through the cytoplasm and into the nucleus where gene expression starts with the transcription of DNA into pre-mRNA. From this start to the final assembly of the biologically-active protein, there are a great number of regulatory mechanisms that can affect gene expression and various signalling pathways can participate in many of these mechanisms, both at transcriptional and posttranscriptional levels. Thus cytokine production is a fast and transient process, initiated and controlled by even faster mechanisms represented by the signalling pathways. The fact that a given signalling pathway regulates the expression of various inflammatory mediators is especially important for therapeutic applications if one considers that targeting expression of a single cytokine may not be particularly effective as the result of compensation of its biologic role by other proinflammatory cytokines.
The MAPKs are a group of conserved cytoplasmic kinases that are organized in modules (MAPKKK → MAPKK → MAPK) sequentially activated by dual phosphorylation at tyrosine/threonine residues. Of the four distinct classes of MAPKs described to date in mammals, p38 (α and β isoforms), c-Jun N-terminal-activated kinases (JNK1-3), and extracellular-activated kinases (ERK1,2) are the most studied. Downstream substrates of MAPKs include a variety of transcription factors, RNA-binding proteins, and other kinases (mitogen-activated protein kinase-activated protein kinases [MAPKAPK]) that are involved in regulation of gene expression by transcriptional, posttranscriptional, translational, and posttranslational mechanisms (Figure 25-2).
The signal transducers and activators of transcription (STAT) family of latent transcription factors is involved in many cytokine signalling pathways,150 especially those that use the gp130 receptor. Many cytokines and growth factors (IFNs, interleukins, epidermal growth factor, growth hormone, erythropoietin, and others) exert their biologic functions through the JAK-STAT signal transduction pathway.224,285 Classically, IFNs and interleukins, cytokines with key roles in regulating the immune response, activate enzymes called Janus kinases (JAK1, JAK2, JAK3, and Tyk2), which are associated with the cytoplasmic portion of the transmembrane receptors.149 Activated JAKs phosphorylate the cytoplasmic domain of the receptor leading the activation of its substrates, especially the proteins known as STATs (STAT1-4, 5a, 5b, and 6). On phosphorylation, STATs may form homodimers or heterodimers, which enables them to enter to the nucleus in which they can regulate gene transcription.224 Although individual STAT proteins may be activated by multiple ligands, certain cytokines preferentially activate particular STATs. IFN-γ preferentially activates STAT1 through JAK1/JAK2, and IL-6 activates STAT3 through JAK1. The JAK-STAT pathway is the signalling target of many cytokines that play important roles in inflammation and thus could also be important in periodontal disease, including IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and TNF-α.32,259,327 STAT3 can be phosphorylated at tyrosine and serine residues. This dual phosphorylation is needed for full activation, even though it is not known if serine phosphorylation of STAT3 has a role in DNA-binding of STAT3.332 Interestingly, deficient STAT3 signalling in innate immune cells has been recently shown to exacerbate P. gingivalis-induced experimental periodontitis in mice by impairing IL-10 signalling and increasing a Th-1 type response characterized by elevated IL-12 and IL-1α.279
NF-κB consists of five related transcription factors that can form multiple homodimers and heterodimers and regulate inducible gene expression in various physiologic contexts.105 Studies on animal and human diseases have indicated that activation of NF-κB is crucial for the expression of various inflammatory genes.158,278 This role of NF-κB for the inflammatory/immune response has been validated by other studies using genetic approaches and chemical inhibitors.6,211,287 The potential therapeutic applications of NF-κB inhibition have been investigated in clinical trials in cancer2 and in animal models of rheumatoid arthritis.208 Collectively, this information suggests that NF-κB signalling should play an important role in periodontal diseases; this concept is supported not only by gene-profiling analysis demonstrating increased expression of both NF-κB and NF-κB–regulated inflammatory genes in diseased periodontal tissues78 but also by studies in vitro.53
However, in spite of a great deal of information available on the regulation and expression of inflammatory cytokines, there are only a few reports on the signalling pathways activated specifically in periodontal disease in vivo. NF-κB activation has been shown to be associated with increased periodontal disease severity.8 Differences in the activation of signalling pathways in two frequently used murine models of experimentally-induced periodontal disease were reported recently: in both the bacterial LPS injection model and the ligature model, p38 and ERK MAPKs, as well as NF-κB, were activated but with different kinetics. On the other hand, activation of JAK-STAT signalling was only observed with the ligature model.100 However, to date, no studies with NF-κB or STAT inhibitors are available in periodontal disease models, despite the important role of these proteins in the regulation of inflammatory genes.
Interestingly, the proteins comprising many of the signalling pathways are highly conserved among different species of organisms, indicating their fundamental role in many essential physiologic processes. Some of these signalling pathways also have a relevant role in diverse pathologic conditions, demonstrating their multivalency. For instance, the p38 MAPK pathway was originally described as critically important to signal stress, inflammatory, and infectious stimuli, but it is also involved in the control of fundamental processes, including cell proliferation,123 differentiation,190 and migration.135 Nevertheless, many reports indicate its relevance and/or potential therapeutic application in disease processes that involve inflammation and immunity, including rheumatoid arthritis,205,210,325 ischemic heart disease,58 allergies,83,280 chronic obstructive pulmonary diseases,212 Alzheimer’s disease,66 and cancer.257
Surprisingly, in spite of evidence indicating a role of p38 MAPK in all of these diseases, there is a relative paucity of information regarding its role in oral inflammation–related conditions, including temporomandibular joint disorders, chronic oral pain, and inflammatory changes of the oral mucosa.240 p38 MAPK is involved in the regulation of expression of proinflammatory cytokines and enzymes induced by inflammatory and infectious signals in vitro, including IL-6,238,239 MMP-13,264,265 and RANKL263 in resident cells of the periodontium such as fibroblasts and osteoblasts. Based on its involvement in the regulation of these cytokines, it is likely that p38 MAPK plays a role in disease progression, since this signalling pathway is not only one of the main downstream effectors of TLR signalling34,37 but also particularly relevant for the activation and development of adaptive immune responses, as demonstrated by its role in T-cell proliferation and cytokine production258,346 and differentiation of immature T cells into Th1 or Th2 effector cells.60 p38 MAPK is also involved in B-cell activation63 and production of cytokines, including IL-1068 and even modulates IL-4–mediated responses in B cells by crosstalk with STAT6.245 This illustrates the multiple roles of this signalling pathway and how modulation of its activity may have multiple effects both on innate and adaptive immunity. Other signalling pathways that have been shown to be activated and involved in regulation of gene expression during inflammation and immune response, such as Notch,10,94 Wnt,109,244 and PI3-kinase261,333 pathways, participate in host-microbe interactions but have not been studied in the context of periodontal disease.
Therapeutic Strategies for Disrupting Host-Cell Signalling in the Treatment of Periodontal Diseases
A variety of treatment strategies have been developed to target the host response to LPS-mediated tissue destruction. MMP inhibitors, such as low dose formulations of doxycycline, have been used in combination with scaling and root planing48 or surgical therapy.98 In addition, high-risk patient populations, such as those with diabetes, have benefited from systemic administration of MMP inhibitors.51,221,270 Encouraging results have also been shown using soluble antagonists of TNF and IL-1 delivered locally to the periodontal tissues in nonhuman primates.17 Other therapeutic strategies that are being explored are aimed at inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of NF-κB and p38 MAP kinase pathways are actively being developed to manage rheumatoid arthritis and inflammatory bone diseases1,158,181 and have been applied in periodontal disease models with some benefits observed.175,260 Using this novel strategy, inflammatory mediators, including proinflammatory cytokines (IL-1, TNF, IL-6), MMPs and others, would be inhibited at the level of cell signalling pathways required for transcription factor activation necessary for inflammatory gene expression or mRNA stability (Figure 25-2). Indeed, targeting RNA-binding proteins that mediate effects on inflammatory cytokines does have therapeutic value in small animal models of periodontal disease progression.241 These therapies may provide the next wave of adjuvant chemotherapeutics that may be used to manage chronic periodontitis.
Science Transfer
A small number of the 500 plus bacterial species found in dental biofilms are related to periodontal destruction. These are gram-negative anaerobic bacteria that reside in the subgingival plaque.
The extent and severity of periodontitis is closely related to the host response to these putative pathologic bacteria. Initially, the host oral bacterial biofilm stimulates the establishment of an innate immune response based on the classical inflammatory cells. With initiation of periodontal disease, dendritic cells signal a more widespread immune reaction (adaptive immune response) that involves not only the B and T lymphocytes, polymorphonuclear leukocytes (PMNs), macrophages, and osteoclasts but also has contributions of signalling molecules and tissue destructive cytokines from resident epithelial cells, fibroblasts, and osteoblasts. The resulting molecular response is complex and interrelated and involves molecular signalling with genetic control of many of the proteins involved. Recent research has unraveled only part of these bioactive networks, and the more that is discovered, the more complicated and diversified are the mechanisms of periodontal destruction.
Most clinicians will have difficulty appreciating all of the signaling pathways and feedback mechanisms that are elucidated, but they should appreciate the importance of these adaptive immune responses in disease progression. New therapies based on regulating tissue destruction by inhibiting such molecules as matrix metalloproteinases (MMPs) with long-term low-dose doxycycline are already in widespread use as supportive therapy for conventional periodontal treatment. Newer approaches soon to be available include antagonists to cytokines and agents to inhibit signalling pathways associated with tissue destructive proteins. Because of the multitudinous molecular mechanisms of tissue destruction, it is unlikely that any one of these approaches will have a singular dramatic therapeutic affect, rather they will be only effectively used in combination with conventional antibacterial treatments such as initial therapy, antimicrobial agents, and periodontal surgery.
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Suggested Reading
Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783-801.
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