CS4-3) Periodontal Pathogenesis PDF

Prof. Dr. TOLGA TOZUM YAKINDOĞU ÜNİVERSİTESİ DİŞHEKİMLİĞİ FAKÜLTESİ Learning outcomes: 1- Will be able to explain the Histologic Stages of Gingivitis 2- Will be able to explain bacterial microbial virulence factors in the pathogenesis of periodontal diseases. 3- Will be able to explain host-derived...

Prof. Dr. TOLGA TOZUM YAKINDOĞU ÜNİVERSİTESİ DİŞHEKİMLİĞİ FAKÜLTESİ Learning outcomes: 1- Will be able to explain the Histologic Stages of Gingivitis 2- Will be able to explain bacterial microbial virulence factors in the pathogenesis of periodontal diseases. 3- Will be able to explain host-derived inflammatory mediators in the pathogenesis of periodontal diseases. 4-Will be able to explain the process of alveolar bone destruction in periodontal diseases. Periodontal Pathogenesis Periodontal disease results from a complex interplay between the subgingival biofilm and the host immune–inflammatory events that develop in the gingival and periodontal tissues in response to the challenge presented by the bacteria. It is generally accepted that gingivitis precedes periodontitis, but it is clear that not all cases of gingivitis progress to periodontitis. With gingivitis, the inflammatory lesion is confined to the gingiva; however, with periodontitis, the inflammatory processes extend to additionally affect the periodontal ligament and the alveolar bone. The net result of these inflammatory changes is the breakdown of the fibers of the periodontal ligament, resulting in clinical loss of attachment togetherwith resorption of the alveolar bone. Histopathology of Periodontal Disease Periodontal disease is a chronic bacterial infection that affects the gingiva and bone that supports the teeth. This chronic inflammatory disease results from the host response to bacteria of dental biofilm. In addition to the presence of periodontopathogens; such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Tannerella forsythia; genetic and environmental factors seem to increase the susceptibility of some individuals in developing this severe inflammatory disease.Periodontal epithelium provides a physical barrier to infection and has an active role in the innate host defense. The epithelium can participate in the infection by signaling further innate and acquired immune responses. Epithelial cells may also respond to bacteria by increasing their proliferation, by altering their cell signaling events, and by changing the cell differentiation and cell death and altering tissue homeostasis. Langerhans cells and dendritic cells are located within the epithelium. They are a connecting link with acquired immunity. During the 1970s and 1980s, bacterial plaque was generally considered to be preeminent as the cause of periodontitis. At that time, it was accepted that poor oral hygiene resulted in increased plaque accumulation, which in turn resulted in periodontal disease. However, this model failed to take into account observations such as the fact that there are many individuals with poor oral hygiene who do not develop advanced periodontal disease and, conversely, that there are unfortunate individuals who, despite good oral hygiene and compliance with periodontal treatment protocols, continue to experience progressive periodontal breakdown and who would be considered to have aggressive periodontitis. These findings were confirmed by the work of Löe and colleagues, who studied Sri Lankan tea laborers who had no access to dental care and who could be divided into three main categories: 1.individuals (≈8% of the population studied) who had a rapid progression of periodontaldisease; 2.those (≈81%) who had a moderate progression of such disease; and 3.those (≈11%) who demonstrated no progression of periodontal disease beyond gingiviti All patients in this population displayed abundant plaque and calculus deposits.The causative role of plaque bacteria is clear in that the bacteria initiate and perpetuate the inflammatory responses that develop in the gingival tissues. However, the major determinant of susceptibility to disease is the nature of the immune–inflammatory responses themselves. It is paradoxical that these defensive processes, which are protective by intent (i.e., to prevent the ingress of the bacteria and their products into the tissues), result in the majority of tissue damage that leads to the clinical manifestations of disease. Periodontal disease is therefore a unique clinical entity. It is not an infection in the classic sense of the word. With most infections, a single infective organism causes the disease , and the identification of that organism provides the basis for the diagnosis. With periodontal disease, a large number of species are identifiable in the periodontal pocket, and many more are as yet unknown because they have not been cultured. It is impossible to conclude that a single species or even a group of species causes periodontal disease. Many of the species that are considered important in periodontal pathogenesis may simply reside in deep pockets because the pocket is a favorable environment in which they can survive. Clinically Healthy Gingival Tissues Even in clinically healthy gingiva, the gingival connective tissue contains at least some inflammatory cells, particularly neutrophils. Neutrophils continually migrate through the connective tissues and pass through the junctional epithelium to enter the sulcus or pocket. This low-grade inflammation occurs in response to the continued presence of bacteria and their products in the gingival crevice.The connective tissue component of the dentogingival unit contains densely packed collagen fiber bundles (a mixture of type I and III collagen fibers) that are arranged in distinct patterns that maintain the functional integrity of the tissues and the tight adapta tion of the soft tissues to the teeth. These include the following: • Dentogingival fibers (extend from the cementum into the free and attached gingiva) • Alveologingival fibers (extend from the alveolar crest into the free and attached gingiva) • Circular fibers (wrap around the tooth, maintain the close adaptation of the free gingiva to the tooth, and interweave with other collagen fiber bundles) • Dentoperiosteal fibers (run from the cementum over the alveolar crest and insert into the alveolar process) • Transseptal fibers (run interdentally from the cementum just apical to the junctional epithelium and over the alveolar crest, where they insert into the cementum of the neighboring tooth). Overt clinical signs of gingivitis (i.e., redness, swelling, and bleeding on probing) do not develop because of several innate and structural defense mechanisms, including the following: • The maintenance of an intact epithelial barrier (the junctional and sulcular epithelium) • The outflow of GCF from the sulcus (dilution effect and flushing action) • The sloughing (slafing) of surface epithelial cells of the junctional and sulcular epithelium • The presence of neutrophils and macrophages in the sulcus that phagocytose bacteria • The presence of antibodies in the GCF (although it is not clear whether these are effective) However, if plaque accumulation increases so that these defense mechanisms are overwhelmed, then inflammation and the classic clinical signs of gingivitis will develop. Although the development of gingivitis in response to the accumulation of plaque is fairly predictable, research has identified that a spectrum of responses may be observed, with some individuals developing marked gingival inflammation for a given plaque challenge and others developing minimal gingival inflammation. These observations underscore the importance of variations in host responses between individuals in terms of gingival inflammatory responses. Furthermore, many individuals may never develop periodontitis, despite having widespread gingivitis. Histopathology of Gingivitis and Periodontitis The development of gingivitis is very clearly observed from a clinical perspective. In addition, the changes that occur within the tissues are very obvious when examined under a microscope. In broad terms, there is infiltration of the connective tissues by numerous defense cells, particularly neutrophils, macrophages, plasma cells, and lymphocytes. As a result of the accumulation of these defense cells and the extracellular release of their destructive enzymes, there is disruption of the normal anatomy of the connective tissues that results in collagen depletion and subsequent proliferation of the junctional epithelium. Vasodilation and increased vascular permeability lead to increased leakage of fluid out of the vessels and facilitate the passage of defense cells from the vasculature into the tissues, resulting in enlargement of the tissues, which appear erythematous and edematous (i.e., the clinical appearance of gingivitis). These changes are all reversible if the bacterial challenge is substantially reduced by improved oral hygiene. The Initial Lesion. The initial lesion is typically said to develop within 2 to 4 days of the accumulation of plaque at a site that was otherwise free of plaque and at which there was no inflammation evident microscopically. However, this situation is probably never encountered in reality, and the gingival tissues always have characteristics of a low-grade chronic inflammatory response as a result of the continual presence of the subgingival biofilm. In other words, the initial lesion corresponds with the histologic picture that is evident in clinically healthy gingival tissues. This low-grade inflammation is characterized by dilation of the vascular network and increased vascular permeability, thus permitting the neutrophils and monocytes from the gingival vasculature to migrate through the connective tissues toward the source of the chemotactic stimulus: the bacterial products in the gingival sulcus. The upregulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin in the gingival vasculature facilitates the migration of neutrophils from the capillaries into the connective tissues. The increased leakage of fluid from the vessels increases the hydrostatic pressure in the local microcirculation, and, as a result, GCF flow increases. Increased GCF flow has the effect of diluting bacterial products, and it also potentially has a flushing action to remove bacteria and their products from the crevice. However, given the nature of the bacterial biofilm, it is likely that only planktonic (free-floating) bacteria are removed in this way. The Early Lesion. The early lesion develops after about 1 week of continued plaque accumulation and corresponds with the early clinical signs of gingivitis. The gingiva are erythematous in appearance as a result of the proliferation of capillaries, the opening up of microvascular beds, and continued vasodilation. Increasing vascular permeability leads to increased GCF flow, and transmigrating neutrophils increase significantly in number. The predominant infiltrating cell types are neutrophils and lymphocytes (primarily thymic lymphocytes [T cells]),136 and the neutrophils migrate through the tissues to the sulcus and phagocytose bacteria. Fibroblasts degenerate, primarily via apoptosis (programmed cell death), which increases the space available for infiltrating leukocytes. The Established Lesion. The established lesion roughly corresponds with what clinicians would refer to as “chronic gingivitis.” The progression from the early lesion to the established lesion depends on many factors, including the plaque challenge (the composition and quantity of the biofilm), host susceptibility factors, and risk factors (both local and systemic). In the initial work by Page and Schroeder, the established lesion was defined as being dominated by plasma cells. In human studies, reports have suggested that plasma cells predominate in established gingivitis in older subjects, whereas lymphocytes predominate in younger individuals, although the relevance of these findings is not clear. What is clear from all of the studies is that there is a significant inflammatory cell infiltrate in established gingivitis that occupies a considerable volume of the inflamed connective tissues. Large numbers of infiltrating cells can be identified adjacent and lateral to the junctional and sulcular epithelium, around blood vessels, and between collagen fiber bundles. The Advanced Lesion. The advanced lesion marks the transition from gingivitis to periodontitis. This transition is determined by many factors, the relative importance of which is currently unknown but which includes the bacterial challenge (both the composition and the quantity of the biofilm), the host inflammatory response, and susceptibility factors, including environmental and genetic risk factors. Histologic examination reveals continued evidence of collagen destruction that extends into the periodontal ligament and the alveolar bone. Neutrophils predominate in the pocket epithelium and the periodontal pocket, and plasma cells dominate in the connective tissues. The junctional epithelium migrates apically along the root surface into the collagen-depleted areas to maintain an intact epithelial barrier. Osteoclastic bone resorption commences, and the bone retreats from the advancing inflammatory front as a defense mechanism to the prevent spread of bacteria into the bone. Inflammatory Responses in the Periodontium Now that the histopathology of gingivitis and periodontitis has been reviewed, it is important to consider some of the specific molecules that signal tissue damage as the inflammatory response develops. These can be broadly divided into two main groups: those derived from the subgingival microbiota (i.e., microbial virulence factors) and those derived from the host immune–inflammatory response. Microbial Virulence Factors The subgingival biofilm initiates and perpetuates inflammatory responses in the gingival and periodontal tissues. The subgingival bacteria also contribute directly to tissue damage by the release of noxious substances, but their primary importance in periodontal pathogenesis is that of activating immune–inflammatory responses that in turn result in tissue damage, which may well be beneficial to the bacteria located within the periodontal pocket by providing nutrient sources. Microbial virulence factors that are important in these processes are now discussed in turn. Lipopolysaccharide. Lipopolysaccharides (LPSs) are large molecules composed of a lipid component (lipid A) and a polysaccharide component. They are found in the outer membrane of gram-negative bacteria, they act as endotoxins (LPSs are frequently referred to as endotoxins), and they elicit strong immune responses in animals. LPSs are highly conserved in gram-negative bacterial species, which reflects their importance in maintaining the structural integrity of the bacterial cells. Immune systems in animals have evolved to recognize LPS via Toll-like receptors (TLRs), a family of cell surface molecules that are highly conserved in animal species ranging from Drosophila (a genus of fruit flies) to humans, thereby reflecting their importance in innate immune responses. TLRs are also present in lower animals and are in fact more varied than in higher species. TLRs are cell surfacereceptors that recognize microbe-associated molecular patterns (MAMPs), which are conserved molecular structures located on diverse pathogens. TLR-4 recognizes LPSs from gram-negative bacteria and functions as part of a complex of cell surface molecules, including CD14 and MD-2 (also known as lymphocyte antigen). The interaction of this CD14/TLR-4/MD-2 complex with LPSs triggers a series of intracellular events, the net result of which is the increased production of inflammatory mediators (most notably cytokines) and the differentiation of immune cells (e.g., dendritic cells) for the development of effective immune responses against the pathogens. It is particularly interesting to the periodontist that the pathogen Porphyromonas gingivalis has an atypical form of LPSs that are recognized by both TLR-2 and TLR-4. Bacterial Enzymes and Noxious Products. Plaque bacteria produce a number of metabolic waste products that contribute directly to tissue damage. These include noxious agents such as ammonia (NH3) and hydrogen sulfide (H2S) as well as short-chain carboxylic acids such as butyric acid and propionic acid. These acids are detectable in GCF and found in increasing concentrations as the severity of periodontal disease increases. These substances have profound effects on host cells (e.g., butyric acid induces apoptosis in T cells, B cells, fibroblasts, and gingival epithelial cells). The short-chain fatty acids may aid P. gingivalis infection through tissue destruction, and they may also create a nutrient supply for the organism by increasing bleeding into the periodontal pocket. Microbial Invasion. Microbial invasion of the periodontal tissues has long been a contentious topic. In histologic specimens, bacteria (including cocci, filaments, and rods) have been identified in the intercellular spaces of the epithelium.Periodontal pathogens such as P. gingivalis and Aggregatibacter actinomycetemcomitans have been reported to invade the gingival tissues,including the connective tissues. Fusobacterium nucleatum can invade oral epithelial cells, and bacteria that routinely invade host cells may facilitate the entry of noninvasive bacteria by coaggregating with them Fimbriae. The fimbriae of certain bacterial species, particularly P. gingivalis, may also play a role in periodontal pathogenesis. P. gingivalis fimbriae stimulate immune responses, such as IL-6 secretion, and the major fimbrial structural component of P. gingivalis, FimA, has been shown to stimulate nuclear factor (NF)- κβ and IL-8 in a gingival epithelial cell line via TLR-2. Mono- cytes are also stimulated by P. gingivalis FimA, secreting IL-6, IL-8, and TNF-α.P. gingivalis fimbriae also interact with complement receptor-3 (CR-3) to activate intracellular signaling pathways that inhibit IL-12 production mediated by TLR-2 signalling. This may be of clinical relevance, because IL-12 is important in the activation of natural killer (NK) cells and CD8+ cytotoxic T cells, which themselves may be important in killing P. gingivalis–infected host cells, such as epithelial cells. Indeed, the blockade of the CR-3 receptor promotes IL-12–mediated clearance of P. gingivalis and negates its virulence.66 Bacterial fimbriae are therefore important for modifying and stimulating immune responses in the periodontium. Bacterial Deoxyribonucleic Acid and Extracellular Deoxyribonucleic Acid. Bacterial deoxyribonucleic acid (DNA) stimulates immune cells via TLR-9, which recognizes hypomethylated CpG regions of the DNA. CpG sites are regions of DNA at which a cytosine nucleotide is found next to a guanine nucleotide (separated by a phosphate molecule, which links the C and G nucleotides together, hence “CpG”). Extracellular DNA (eDNA) is likely to play a role in the development and structure of the biofilms formed by oral bacteria, and it has been identified as an important component of the matrix in a number of bacterial biofilms. Host-Derived Inflammatory Mediators The inflammatory and immune processes that develop in the periodontal tissues in response to the long-term presence of the subgingival biofilm are protective by intent but result in considerable tissue damage. This has sometimes been referred to as bystander damage, which denotes that the host response is mainly responsible for the tissue damage that occurs, thereby leading to the clinical signs and symptoms of periodontal disease. It is paradoxical that the host response causes most of the tissue damage, although this is by no means unique to periodontal disease. For example, the tissue damage that occurs in the joints in patients with rheumatoid arthritis results from prolonged and excessive inflammatory responses, and it is characterized by the increased production of many of the cytokines that are known to be important in periodontal pathogenesis. In the case of rheumatoid arthritis, the initiating factor is an autoimmune response to structural components of the joint; in periodontitis, the initiating factor is the subgingival biofilm. In both cases, however, the destructive inflammatory events are remarkably similar, although the pathogenesis varies as a result of the different anatomy. Cytokines. Cytokines play a fundamental role in inflammation, and they are key inflammatory mediators in periodontal disease.They are soluble proteins, and they act as messengers to transmit signals from one cell to another. Cytokines bind to specific receptors on target cells, and they initiate intracellular signaling cascades that result in phenotypic changes in the cell via altered gene regulation. Cytokines are effective in very low concentrations, they are produced transiently in the tissues, and they primarily act locally in the tissues in which they are produced. Cytokines are able to induce their own expression in either an autocrine or paracrine fashion, and they have pleiotropic effects (i.e., multiple biologic activities) on a large number of cell types. Prostaglandins. The prostaglandins (PGs) are a group of lipid compounds derived from arachidonic acid, a polyunsaturated fatty acid found in the plasma membrane of most cells. Arachidonic acid is metabolized by cyclooxygenase-1 and -2 (COX-1 and COX-2) to generate a series of related compounds called the prostanoids, which includes the PGs, the thromboxanes, and the prostacyclins. PGs are important mediators of inflammation, particularly prostaglandin E2 (PGE2), which results in vasodilation and induces cytokine production by a variety of cell types. COX-2 is upregulated by IL-1β, TNF-α, and bacterial LPS, resulting in increased production of PGE2 in inflamed tissues. PGE2 is produced by various types of cells and most significantly in the periodontium by macrophages and fibroblasts. PGE2 results in the induction of MMPs and osteoclastic bone resorption, and it has a major role in contributing to the tissue damage that characterizes periodontitis. Matrix Metalloproteinases. MMPs are a family of proteolytic enzymes that degrade extracellular matrix molecules such as collagen, gelatin, and elastin. They are produced by a variety of cell types, including neutrophils, macrophages, fibroblasts, epithelial cells, osteoblasts, and osteoclasts.. The nomenclature of MMPs has been based on the perception that each enzyme has its own specific substrate; for example, MMP-8 and MMP-1 are bot collagenases (i.e., they break down collagen). However, it is now appreciated that MMPs usually degrade multiple substrates, with significant substrate overlap between individual MMPs. The substrate-based classification is still used, however, and MMPs can be divided into collagenases, gelatinases/type IV collagenases, stromelysins, matrilysins, membrane-type metalloproteinases, and others. Role of Specific Inflammatory Mediators in Periodontal Disease Interleukin-1 Family Cytokines. The IL-1 family of cytokines comprises at least 11 members, including IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-33.IL-1β plays a key role in inflammation and immunity; it is closely linked to the innate immune response, and it induces the synthesis and secretion of other mediators that contribute to inflammatory changes and tissue damage. For example, IL-1β stimulates the synthesis of PGE2, platelet-activating factor, and nitrous oxide, thereby resulting in vascular changes associated with inflammation and increasing blood flow to the site of infection or tissue injury. IL-1β is mainly produced by monocytes, macrophages, and neutrophils and also by other cell types such as fibroblasts, keratinocytes, epithelial cells, B cells, and osteocytes.IL-1β increases the expression of ICAM-1 on endothelial cells and stimulates the secretion of the chemokine CXCL8 (which is IL-8), thereby stimulating and facilitating the infiltration of neutrophils into the affected tissues. IL-1β also synergizes with other proinflammatory cytokines and PGE2 to induce bone resorption. IL-1β has a role in adaptive immunity; it regulates the development of antigen-presenting cells (APCs) (e.g., dendritic cells), stimulates IL-6 secretion by macrophages (which in turn activates B cells), and has been shown to enhance the antigen-mediated stimulation of T cells.IL-18 interacts with IL-1β and shares many of the proinflammatory effects of IL-1β.It is mainly produced by stimulated monocytes and macrophages. There is increasing evidence to suggest that IL-18 plays a significant role in inflammation and immunity. IL-18 results in pro-inflammatory responses, including the activation of neutrophils. It is a chemoattractant for T cells,and it interacts with IL-12 and IL-15 to induce interferon gamma (IFN-γ), thereby inducing T-helper (Th1) cells, which activate cellmediated immunity.199 Interestingly, in the absence of IL-12, IL-18 induces IL-4 and a Th2 response, which regulates humoral (antibody-mediated) immunity. There is very limited direct evidence for a role of IL-18 in periodontal pathogenesis. Oral epithelial cells secrete IL-18 in response to stimulation with LPS, and a correlation between GCF IL-18 levels and sulcus depth has been reported. IL-18 levels have been reported to be higher than those of IL-1β in patients with periodontitis, thereby suggesting that IL-18—along with IL-1β—is predominant in periodontitis lesions. Because IL-18 has the ability to induce either Th1 or Th2 differentiation, it is likely to play an important role in periodontal disease pathogenesis. Other Interleukin-1 Family Cytokines. Six new members of the IL-1 family (IL-1F) of cytokines have been identified on the basis of their sequence homology, structure, gene location, and receptor binding. Several of these cytokines were identified b different groups, who gave them a variety of names, and proposals were suggested for renaming all of the IL-1F cytokines in a more consistent manner, as indicated . Our knowledge of the role of these cytokines in inflammation and immunity is very limited at present, and some of these cytokines may be evolution arily redundant. IL-1F6, IL-1F8, and IL-1F are potential agonists (stimulating proinflammatory responses),19,182 whereas IL-1F5 and IL-1F10 are potential antagonists. IL-1F7 appears to have anti-inflammatory action. It has five splice variants and one isoform, IL-1F7b, which is highly expressed by monocytes and upregulated by LPS. An intracellular mode of action has been suggested for IL-1F7b; it translocates to the nucleus of macrophages, and it may act as a transcriptional modulator by reducing the production of LPS-stimulated proinflammatory cytokines, thussupporting an anti-inflammatory role for this cytokine Tumor Necrosis Factor-α. TNF-α is a key inflammatory mediator in periodontal disease, and it shares many of the cellular actions of IL-1β. It plays a fundamental role in immune responses, it increases neutrophil activity, and it mediates cell and tissue turnover by inducing MMP secretion. TNF-α stimulates the development of osteoclasts and limits tissue repair via the induction of apoptosis in fibroblasts. TNF-α is secreted by activated macrophages as well as by other cell types, particularly in response to bacterial LPS. The proinflammatory effects of TNF-α include the stimulation of endothelial cells to express selectins that facilitate leukocyte recruitment, the activation of macrophage IL-1β production, and the induction of PGE2 by macrophages and gingival fibroblasts.134 TNF-α—although it possesses similar activity to IL-1β—has a less potent effect on osteoclasts, and it is present at lower levels in inflamed gingival tissues than IL-1β.GCF levels of TNF-α increase as gingival inflammation develops, and higher levels are found in individuals with periodontitis. The importance of TNF-α (and IL-1β) in periodontal pathogenesis is unquestioned, and it has particularly been highlighted by studies showing that the application of antagonists to IL-1β and TNF-α resulted in an 80% reduction in recruitment of inflammatory cells in proximity to the alveolar bone and a 60% reduction in bone loss. Interleukin-6 and Related Cytokines. The cytokines in this group—which include IL-6, IL-11, leukemia-inhibitory factor (LIF), and oncostatin M—share common signaling pathways via signal transducers glycoprotein (gp) 130.74 IL-6 is the most extensively studied of this group, and it has pleiotropic proinflammatory properties.IL-6 secretion is stimulated by cytokines such as IL-1β and TNF-α, and it is produced by a range of immune cells (e.g., T cells, B cells, macrophages, dendritic cells) as well as resident cells (e.g., keratinocytes, endothelial cells, fibroblasts).IL-6 is also secreted by osteoblasts, and it stimulates bone resorption and the development of osteoclasts.81,94 IL-6 is elevated in the cells, tissues, and GCF of patients with periodontal disease., IL-6 may have an influence on monocyte differentiation into osteoclasts and a role in bone resorption in patients with periodontal disease.IL-6 also has a key role in regulating the proliferation and differentiation of B cells and T cells, particularly the Th17 subset.IL-6 therefore has an important role in periodontal pathogenesis, although it is less than that of IL-1β or TNF-α. Prostaglandin E2. The cells primarily responsible for PGE2 production in the periodontium are macrophages and fibroblasts. PGE2 levels are increased in the tissues and the GCF at sites undergoing periodontal attachment loss. PGE2 induces the secretion of MMPs as well as osteoclastic bone resorption, and it contributes significantly to the alveolar bone loss seen with periodontitis. PGE2 release from monocytes from patients with severe or aggressive periodontitis is greater than that from monocytes from patients who are periodontally healthy. A large body of evidence has demonstrated the importance of PGE2 in periodontal pathogenesis, and, given that prostaglandins are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), researchers have investigated the use of NSAIDs as potential host–response modulators in the management of periodontal disease. However, daily administration for extended periods is necessary for the periodontal benefits to become apparent, and NSAIDs are associated with significant unwanted side effects, including gastrointestinal problems, hemorrhage (from impaired platelet aggregation resulting from inhibition of thromboxane formation), and renal and hepatic impairment. NSAIDs are therefore not indicated as adjunctive treatments for the management of periodontitis. Matrix Metalloproteinases. MMPs are a family of zincdependent enzymes that are capable of degrading extracellular matrix molecules, including collagens. MMPs play a key role in periodontal tissue destruction and are secreted by the majority of cell types in the periodontium, including fibroblasts, keratinocytes, endothelial cells, osteoclasts, neutrophils, and macrophages. In healthy tissues, MMPs are mainly produced by fibroblasts, which produce MMP-1 (also known as collagenase-1), and these have a role in the maintenance of the periodontal connective tissues. The transcription of genes coding for MMPs is upregulated by cytokines such as IL-1β and TNF-α. MMP activity is regulated by specific endogenous TIMPs and serum glycoproteins such as α-macroglobulins, which form complexes with active MMPs and their latent precursors. TIMPs are produced by fibroblasts, macrophages, keratinocytes, and endothelial cells; they are specific inhibitors that bind to MMPs in a 1 :1 stoichiometry. MMPs are also produced by some periodontal pathogens, such as A. actinomycetemcomitans and P. gingivalis, but the relative contribution of these bacterially derived MMPs to periodontal pathogenesis is small. The great majority of MMP activity in the periodontal tissues is derived from infiltrating inflammatory cells. Chemokines. Chemokines are cytokine-like molecules that are characterized by their chemotactic activity. This activity gave rise to the term chemokine (i.e., they are chemotactic cytokines). Chemokines orchestrate leukocyte recruitment in physiologic and pathologic conditions, so they are important for periodontal pathogenesis, which results in the chemotactic migration of neutrophils through the periodontal tissues toward the site of the bacterial challenge in the periodontal pocket. Chemokines play a key role in neutrophil recruitment and the recruitment of other adaptive and innate immune cells to the site of immune and inflammatory responses. The chemokines are divided into two subfamilies according to structural similarity: the CC subfamily and the CXC subfamily. The chemokine CXCL8, which is more familiarly known as IL-8, has been demonstrated to be localized in the gingival tissues in areas of plaque accumulation and in the presence of neutrophilic infiltration,and it has also been found in GCF. Anti-inflammatory Cytokines. The balance between proinflammatory and anti-inflammatory events is crucial for determining disease progression, and it is now clear that individual cytokines do not act in isolation but rather as part of complex networks of mediators that have different functional activities. Antiinflammatory cytokines include IL-10, TGF-β, IL-1Ra, IL-1F5, and possibly IL-1F10. The IL-10 family of cytokines has multiple pleiotropic effects and possesses immunosuppressive properties.32,34 IL-10 is produced by Treg cells, monocytes, and B cells, and it suppresses cytokine secretion by Th1 cells, Th2 cells, monocytes, and macrophages. The role of IL-10 in periodontal disease has been minimally studied, but animal models support that IL-10 downregulates inflammatory responses. For example, IL-10 knockout mice are more susceptible to alveolar bone loss than wild-type mice.IL-10 is also present in GCF and periodontal tissues. Linking Pathogenesis to Clinical Signs of Disease Advanced forms of periodontal disease are characterized by the distressing symptoms of tooth mobility and tooth migration. These result from the loss of attachment between the tooth and its supporting tissues after the breakdown of the inserting fibers of the periodontal ligament and the resorption of alveolar bone. Having reviewed the histopathology and the inflammatory processes that develop in the periodontal tissues as a result of prolonged accumulation of dental plaque, it is now necessary to link these changes to the structural damage that occurs in the periodontium, thereb leading to the well-defined signs of disease. It is important to note that even clinically healthy tissues demonstrate signs of inflammation when histologic sections are examined. For example, transmigrating neutrophils are evident in clinically healthy gingival tissues moving toward the sulcus for the purpose of eliminating bacteria. If the inflammation becomes more extensive, for example, because of an increase in the bacterial challenge, then vasodilation and increased vascular permeability lead to edema of the tissues as well as erythema, thereby causing gingival swelling, a slight deepening of the sulcus, and further compromising plaque removal. The increased infiltration of inflammatory cells (particularly neutrophils) and the breakdown of collagen result in the development of collagen-depleted areas below the epithelium; as a result, the epithelium proliferates to maintai tissue integrity. Alveolar Bone Resorption As the advancing inflammatory front approaches the alveolar bone, osteoclastic bone resorption commences. This is a protective mechanism to prevent bacterial invasion of the bone, but it ultimately leads to tooth mobility and even tooth loss. The resorption of alveolar bone occurs simultaneously with the breakdown of the periodontal ligament in the inflamed periodontal tissues. There are two critical factors that determine whether bone loss occurs: (1) the concentration of inflammatory mediators in the gingival tissues must be sufficient to activate the pathways that lead to bone resorption; and (2) the inflammatory mediators must penetrate to within a critical distance of the alveolar bone. Receptor Activator of Nuclear Factor-κβ Ligand and Osteoprotegerin A key system for controlling bone turnover is the receptor activator of nuclear factor-βB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. RANK is a cell surface receptor expressed by osteoclast progenitor cells as well as by mature osteoclasts. RANKL is a ligand that binds to RANK and that is expressed by bone marrow stromal cells, osteoblasts, and fibroblasts. The binding of RANKL to RANK results in osteoclast differentiation and activation and thus bone resorption. Another ligand that binds to RANK is OPG, which is produced by bone marrow stromal cells, osteoblasts, and fibroblasts. Thus, RANKL and OPG are both cytokines that bind to RANK and that result in cellular responses. However, although RANKL promotes the activation and differentiation of osteoclasts, OPG has the opposite effect, instead inhibiting the differentiation of osteoclasts. The balance between OPG and RANKL activity can therefore drive bone resorption or bone formation. IL-1β and TNF-α regulate the expression of RANKL and OPG, and T cells express RANKL, which binds directly to RANK on the surfaces of osteoclast progenitors and osteoclasts, thereby resulting in cell activation and differentiation to form mature osteoclasts. In individuals with periodontitis, elevated levels of proinflammatory cytokines (e.g., IL-1β, TNF-α) and increasing numbers of infiltrating T cells result in the activation of osteoclasts via RANK, which results in alveolar bone loss. It has been reported that levels of RANKL are higher and that levels of OPG are lower in sites with active periodontal breakdown as compared with sites with healthy gingiva. In addition, GCF RANKL:OPG ratios are higher with periodontitis than in healthy tissue. It is clear that alterations in the relative levels of these key regulators of osteoclasts play a key role in the bone loss that characterizes periodontal disease. Resolution of Inflammation Inflammation is an important defense mechanism to combat the threat of bacterial infection, but inflammation also results in tissue damage associated with the development and progression of most chronic diseases associated with aging, including periodontal disease. It is becoming evident that the resolution of inflammation (i.e., “turning off” inflammation) is an active process that is regulated by specific mechanisms that restore homeostasis. It is possible that controlling or augmenting these mechanisms may lead to the development of new treatment strategies for managing chronic diseases such as periodontitis, and the mechanisms that regulate resolution of inflammation have begun to be identified. Lipoxins The lipoxins include lipoxin A4 (LXA4) and lipoxin B4 (LXB4), and the appearance of these molecules signals the resolution of inflammation. Lipoxins are lipoxygenase (LO)-derived eicosanoids that are generated from arachidonic acid. They are highly potent, they possess biologic activity at very low concentrations, and they inhibit neutrophil recruitment, chemotaxis, and adhesion. Lipoxins also signal macrophages to phagocytose the remnants of apoptotic cell at sites of inflammation without generating an inflammatory response. Pro-inflammatory cytokines (e.g., IL-1β) released during acute inflammation can induce the expression of lipoxins, which promote the resolution of the inflammatory response. Resolvins and Protectins Resolvins (i.e., resolution phase interaction products) are derived from the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid; they are classified as the E series resolvins (RvE) and the D series resolvins (RvD), respectively.Resolvins inhibit neutrophil infiltration and transmigration as well as the production of pro-inflammatory mediators, and they have potent antiinflammatory and immunoregulatory effects. Resolvins are highly potent and have been shown to reduce neutrophil transmigration by around 50% at concentrations of as low as 10 nM.Protectins are also derived from docosahexaenoic acid. They are produced by glial cells, and they reduce cytokine expression.They also inhibit neutrophil infiltration, and they have been reported to reduce retinal injury and stroke damage. Immune Responses in Periodontal Pathogenesis The immune system is essential for the maintenance of periodontal health, and it is central to the host response to periodontal pathogens. However, if the immune response is dysregulated, inappropriate, persistent, or excessive in some way, then damaging chronic inflammatory responses such as those observed with periodontal disease can ensue. The immune response to plaque bacteria involves the integration at the molecular, cellular, and organ level of elements that are often categorized as being part of the innate immune system or the adaptive immune system. Furthermore, host responses in periodontal disease (and other major human diseases) were until recently represented as a linear progression from the host’s recognition of microbial pathogens to innate immune responses dominated by the action of phagocytic neutrophils and culminating in the establishment of adaptive immune responses led by antigen-specific effector functions (e.g., cytotoxic T cells, antibodies). It is now widely appreciated that immune responses are complex biologic networks in which pathogen recognition, innate immunity, and adaptive immunity are integrated and mutually dependent. This complex network is flexible and dynamic, with aspects of positive and negative regulation as well as feedback control; signals are amplified and broadcast, which leads to diverse effector functions. Furthermore, the immune system is integrated with other systems and processes, including the nervous system, hematopoiesis, and hemostasis as well as elements of tissue repair and regeneration. Innate Immunity Defenses against infection include a wide range of mechanical, chemical, and microbiologic barriers that prevent pathogens from invading the cells and tissues of the body. Saliva, GCF, and the epithelial keratinocytes of the oral mucosa all protect the underlying tissues of the oral cavity and in particular the periodontium. The commensal microbiota (e.g., in dental plaque) may also be important for providing protection against infection by pathogenic microorganisms through effective competition for resources and ecologic niches and also by stimulating protective immune responses. The complex microanatomy of the periodontium, including the diversity of specialized epithelial tissues, presents many interesting challenges for the study of the immunopathogenesis of periodontal disease. Saliva. Saliva that is secreted from the three major salivary glands (i.e., parotid, submandibular, and sublingual) as well as from the numerous minor salivary glands has an important role in the maintenance of oral and dental health. The action of shear forces associated with saliva flow is important for preventing the attachment of bacteria to the dentition and the oral mucosal surfaces. Human saliva also contains numerous molecular components that contribute to host defenses against bacterial colonization and periodontal disease.. These components include molecules that nonspecifically inhibit the formation of the plaque biofilm by inhibiting adherence to oral surfaces and promoting agglutination (e.g., mucins), those that inhibit specific virulence factors (e.g., histatins that neutralize LPS), and those that inhibit bacterial cell growth (e.g., lactoferrin) and that may induce cell death. Saliva also contains specific immunoglobulin A (IgA) antibodies to periodontal pathogens that target specific antigens and that inhibit bacterial adherence. Patients with periodontal disease have elevated levels of specific IgA as well as of IgG and IgM, which are antibodies to periodontal pathogens. However, tooth surfaces coated with a salivary pellicle can provide attachment opportunities for plaque bacteria; thus, P. gingivalis can attach to the salivary pellicle via fimbriae. Epithelial Tissues. The epithelial tissues play a key role in host defense, because they are the main site of the initial interactions between plaque bacteria and the host, and they are also the site of the invasion of microbial pathogens. The keratinized epithelium of the sulcular and gingival epithelial tissues provides protection for the underlying periodontal tissue in addition to acting as a barrier against bacteria and their products. By contrast, the unique microanatomic structure of the junctional epithelium has significant intercellular spaces; it is not keratinized, and it exhibits a higher cellular turnover rate. These properties render the junctional epithelium permeable, thereby allowing for the inward movement of microbes and their products and the outward movement of GCF and the cells and molecules of innate immunity. Furthermore, the spaces between the cells of the junctional epithelium widen with inflammation, which results in increased GCF flow. Gingival Crevicular Fluid. GCF originates from the postcapillary venules of the gingival plexus. It has a flushing action in the gingival crevice, but it also likely functions to bring the blood components (e.g., neutrophils, antibodies, complement components) of the host defenses into the sulcus.65 The flow of GCF increases in inflammation, and neutrophils are an especially important component of GCF in periodontal health and disease. Pathogen Recognition and Activation of Cellular Innate Responses. If plaque bacteria and their products penetrate the periodontal tissues, specialized “sentinel cells” of the immune system recognize their presence and signal protective immune responses. These cells include macrophages and dendritic cells, which express a range of pattern recognition receptors (PRRs) that interact with specific molecular structures on microorganisms called MAMPs. The activation of PRRs activates innate immune responses to provide immediate protection, and adaptive immunity is also activated with the aim of establishing a sustained antigen-specific defense. Excessive and inappropriate or dysregulated immune responses lead to chronic inflammation and the concomitant tissue destruction associated with periodontal disease. A glossary of terms relevant to periodontal immunobiology. The best studied of the signaling systems involved in the recognition of plaque bacteria is the interaction of bacterial LPS with TLRs: P. gingivalis, A. actinomycetemcomitans, and F. nucleatum all possess LPS molecules that interact with TLR-4 to activate myeloid immune cells. However, individual species of plaque bacteria have a wide variety of MAMPs, which may interact with PRRs. For example, P. gingivalis LPS signals via TLRs (predomi nantly TLR-2), and fimbriae, proteases, and DNA from P. gingivalis are all recognized by host cells through interaction with specific PRRs. A number of nonimmune cells in the periodontium (e.g., epithelial cells, fibroblasts) also express PRRs and may recognize and respond to MAMPs from plaque bacteria. Although the signaling pathways activated by PRRs may be diverse, in general terms, they converge to elicit similar host cell responses in the form of the upregulation of cytokine secretion and, in the case of APCs such as dendritic cells, cell differentiation that leads to enhanced signaling of the adaptive immune response. Dendritic cells also have C-type lectin receptors (e.g., mannose receptor, langerin, DC-SIGN) that recognize glycans on pathogens. However, the role of these interactions in periodontal disease is not known. Neutrophil Function. Although macrophages have phagocytic capabilities, neutrophils are the “professional” phagocytes that are critical to the clearance of bacteria that invade host tissues Neutrophils are present in clinically healthy gingival tissues, and they migrate through the intercellular spaces of the junctional epithelium into the sulcus. This is part of a “low-grade defense” against plaque bacteria, and it is necessary to prevent inflammatio and periodontal tissue damage.The importance of neutrophils to the maintenance of periodontal health is demonstrated clinically by the observations of severe periodontitis in patients with neutrophil defects and the association of periodontitis with experimental immunosuppression in animal models. Small foci of other leukocytes (e.g., lymphocytes, plasma cells, macrophages) are also found in the healthy gingiva. A small proportion (1% to 2%) of the intercellular spaces in healthy junctional epithelium are occupied by neutrophils (and other leukocytes at various stages of differentiation), but this can increase to 30% with even modest inflammation. In the inflammatory state, there are changes to the local vasculature in the gingiva: high endothelial venules develop from the postcapillary venules of the gingival plexus, which facilitates leukocyte emigration and increases the flow of GCF into the pocket. Adaptive Immunity Adaptive immunity has evolved to provide a focused and intense defense against infections that overwhelm innate immune responses. Adaptive immunity is particularly important as ecologic, social, and demographic changes—which alter susceptibility to existing and emerging infective microorganisms—outpace the natural evolution of biologic systems. Furthermore, the development of effective vaccination is, along with the identification of antibiotics, perhaps one of the greatest triumphs of medical science; this success is based on knowledge of the elements and principles of adaptive immunity. Antigen-Presenting Cells. A central element of the activation and function of T cells and B cells is the presentation of antigen by specialized APCs to T cells and the development of specific cytokine milieu that influences the development of T cells with a particular effector function. APCs are sentinel cells in mucosal tissues such as the periodontium. These cells detect and take up microorganisms and their antigens, after which they may migrate to lymph nodes and interact with T cells to present antigen. The periodontium is often compared to other mucosal tissues and the skin in terms of its repertoire of immune cells, and it contains a number of “professional” APCs, including B cells, macrophages, and at least two types of dendritic cells (i.e., dermal dendritic cells and Langerhans cells). These cells naturally express the major histocompatibility complex class II molecules necessary for antigen presentation to T-cell receptors, and they may take up specific antigens and transport them to local lymph nodes, thereby facilitating the activation of specific effector T cells and the generation of an antigen-specific immune response to periodontal pathogens. Although these cells have been identified in periodontal tissues, shown to stimulate antigen specific T-cell responses in experimental systems, and found to generally increase in the presence of periodontitis, their relative contribution to antigen presentation in vivo remains to be determined. The expression of major histocompatibility complex class II molecules may be induced in other cells that are present in the periodontium (e.g., fibroblasts, epithelial cells), which then also take up antigen and present antigen locally in the periodontium. T Cells. There are a number of different subsets of thymic lymphocytes (i.e., T cells) that develop in the bone marrow and thymus and migrate to the peripheral tissues to participate in adaptive immune responses. The expression of the cell surface molecules (CD4 or CD8) or particular T-cell antigen receptors (αβ or γδ) broadly defines functional T-cell subsets that emerge from the thymus. The role of T-cells in periodontal disease has been established through immunohistologic studies of diseased tissues.CD4+ helper T cells are the predominant phenotype in the stable periodontal lesion, and it is thought that alterations in the balance of effector T-cell subsets within the CD4+ population may lead to progression toward a destructive, B-cell–dominated lesion.CD4+T-cell subsets are defined on the basis of their phenotypic characteristics and effector functions. The nature of the APCs, which present antigen to cognate T-cell receptors on T cells, and the presence of specific combinations of cytokines and chemokines influence the nature of the CD4+ T-cell effector subset, which develops from naive T-cells. CD4+ T-cell subsets are defined by the expression of specific transcription factors, and their functional characteristics are associated with their cytokine secretion profile. Antibodies. Specific antibodies are produced in response to an increasing bacterial challenge in periodontal disease and are the endpoint of B-cell activation. Circulating antibodies may be more important than locally produced antibodies. Even so, these generally appear in a high titer but have low biologic activity, so there is some doubt as to their effectiveness. Commensurate with the appearance of antibodies against plaque bacterial antigens is the appearance of differentiated plasma cells that characterize the established lesion in periodontal disease. High levels of antibodies appear in GCF (in addition to those in the circulation), and these are produced locally by plasma cells in periodontal tissues.9 Anti bodies to periodontal pathogens are primarily IgG, with few IgM or IgA types produced.Other P. gingivalis molecules (i.e., fimbriae and hemagglutinin) also act as antigens. Specific antibodies are also generated by serotype-specific carbohydrate antigens (e.g., capsular polysaccharide of P. gingivalis, carbohydrate of A. actinomycetemcomitansLPS). The subclass distribution of antibodies is influenced by cytokines that are derived from monocytes. For example, IgG2 production is regulated by IL-1α, IL-1β, and PGE2 from monocytes as well as by platelet-activating factor from neutrophils. PGE2 and platelet-activating factor indirectly induce Th1 responses and therefore IFN-γ, which stimulates IgG2 production. Individuals with aggressive periodontitis have monocytes that are hyperresponsive to LPS and that produce elevated quantities of PGE2. A. actinomycetemcomitans is commonly associated with aggressive periodontitis, which induces IL-12 production that regulates NK cells and Th1 cells. These cells are a source of IFN-γ, which in turn regulates IgG2. Concept of Host Susceptibility The immune and inflammatory processes that result from the challenge presented by the subgingival biofilm are complex and mediated by a large number of pro-inflammatory and anti-inflammatory cytokines and enzymes that function as a network of mediator with overlapping roles and activity.Immune responses to the bacterial challenge do not occur in isolation but rather take place in the context of other host and environmental factors that influence these responses and thereby determine the progression of disease.A feature of human development and evolution has been that quantitative and qualitative differences exist in immune responses between individuals. Indeed, infectious agents (e.g., bacteria) exert evolutionary selection pressures on the species that they infect. This may be relevant in periodontal disease, and a large number of studies have confirmed that immune cells from patients with periodontal disease secrete higher quantities of proinflammatory cytokines than those who are periodontally healthy.Cytokine profiles are also different in those individuals with immune-mediated diseases as compared with healthy controls. 1-Newman M, Takei H, Klokkevold P, Carranza F. Newman and Carranza (2019); Clinical Periodontology, 13th Ed., Elsevier.