**4. Immune responses in periodontal pathogenesis**

The immune systems are essential for the maintenance of periodontal health and are mainly categorized as innate immune system and the adaptive immune system (**Figure 1**). It is now widely studied that immune responses are complex biologic networks in which pathogen recognition, innate immunity, and adaptive immunity are integrated and mutually dependent [37]. They are also integrated with other systems, including the nervous system, hematopoiesis, and homeostasis as well as elements of tissue repair and regeneration [38] as shown in **Figure 1**.

### **4.1 Innate immunity**

The term "innate immunity" refers to the elements of the immune response that are determined by inherited factors, that have limited specificity, and do not change or improve during an immune response or as a result of previous exposure to a pathogen. Innate immune mechanisms include a number of relatively non-specific mechanisms, including the barrier effect of an intact epithelium, saliva, and GCF (**Figure 1**). 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 [15, 39]. Saliva, secreted from three major salivary glands, plays an important role in preventing the attachment of bacteria to the dentition and the oral mucosal surfaces. These components include molecules that non-specifically 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 lipopolysaccharide (LPS)) and those that inhibit bacterial cell growth (e.g., lactoferrin) and

**Figure 1.** *Immune responses in periodontal pathogenesis.*

that may induce cell death [40, 41]. GCF originating from the postcapillary venules of the gingival plexus carries blood components like neutrophils, antibodies, and complement components which help in host defense mechanism [42].

Saliva, as part of innate immune response, is a key factor in protecting dental enamel, gingiva, and mucosa by flushing microbes and foodstuffs, buffering acids, remineralizing the tooth, providing antimicrobial activity, and permitting selective adhesion of commensal microorganisms to maintain a symbiotic environment in the dental biofilm [43]. The salivary flow rate—high or low—is characteristic of each individual and [44–47] may promote salivary clearance of microbes from the oral cavity. Saliva also contains varying amounts of immunomodulatory interleukin-1β, interleukin-17, and interleukin-23, although it is not known whether they contribute to innate immunity on mucosal surfaces of the oral environment [48].

### *4.1.1 Pathogen recognition and activation of innate response*

The recognition of pathogenic microorganisms and the recruitment of effector cells (e.g., neutrophils) and molecules (e.g., the complement system) are central to effective innate immunity. Innate immune responses are orchestrated by a broad range of cytokines, chemokines, and cell surface receptors, and the stimulation of innate immunity leads to a state of inflammation. When microbes penetrate the periodontal tissues, specialized cells of immune system, macrophages and dendritic cells, express a range of pattern recognition receptors (PRRs) which interact with specific molecular structures on microorganisms called microbe-associated molecular patterns (MAMPs) activating the innate immune responses (**Figure 2**).

### **4.2 Bacterial biofilm formation and development of a host response**

Biofilms have been defined as "organized microbial communities characterized by a first group of colonizers being irreversibly adhered to a substrate or interphase in a wet media and the rest being embedded in a matrix composed of extracellular polysaccharides produced by the bacteria." The tooth surface provides a non-shedding hard surface where bacteria can adhere and form complex biofilms [8, 49].

The combination of natural host defense mechanisms and oral hygiene practices of individuals helps to have a balanced coexistence of oral microbiota in a healthy oral cavity which can be disturbed by either quantitative (higher bacterial load) or qualitative (growth of pathogenic species) changes in the biofilm leading to early stages of gingivitis [49].

**17**

*Pathogenesis of Gingivitis*

sulcus [8].

*DOI: http://dx.doi.org/10.5772/intechopen.91614*

**4.3 Innate immune response and gingivitis**

and macrophages (**Figure 2**).

**4.4 Activation of adaptive immunity**

innate immune system in early gingivitis lesions [13].

The epithelial attachment of tooth is a highly specialized structure where the junctional epithelial cells strongly attach to the tooth surface by a basal membrane, and hemidesmosomes providing the antibacterial defense mechanism by the high regeneration and desquamation rate and the continuous flow of gingival fluid through the gingival sulcus. The cells of the junctional epithelium with antibacterial proteins like human β-defensin 1 and chemokines along with intercellular adhesion molecule-1 (ICAM-1) and IL-8 help in the migration of PMN toward the gingival

The protective function of the gingival epithelium is enhanced by keratinization, which helps resist abrasion. The gingival epithelium, as an innate immune barrier, is formed by interconnecting keratinocytes bridged one to another by cell adhesion molecules (CAMs) [50] which include integrins, mediating cell interactions with the extracellular matrix and basement membranes and contributing to cell-cell adhesion [51–53], as well as cadherins, which form tight contacts between cells [54]. The CAMs of the multilayered syncytium are susceptible to digestion by gingipains from *Porphyromonas gingivalis*, which could increase tissue permeability [55–58].

Innate immunity is the first line of defense and the cells responsible for the innate immune response are mainly PMN, macrophages, and dendritic cells. Polymorphonuclear leukocytes (PMNs) are the first and predominant cells of the

The biofilm microbes on the tooth surfaces are recognized by the cells from the innate immunity through certain molecular patterns called pathogen-associated molecular patterns (PAMPs) which include lipopolysaccharide (LPS), peptidoglycans and lipoteichoic acids, N-formylmethionine, and lipoproteins. These molecules are recognized by pattern recognition receptors (PRRs) on the surface of PMNL

The two major families of PRRs that have been most extensively studied in the periodontium are the Toll-like receptor (TLRs) and the Nod-like receptors (NLRs) [59]. Toll-like receptors are unique receptors that recognize molecules that are broadly shared by microorganisms but are distinguishable from host molecules and can detect multiple pathogen-associated molecular patterns, including lipopolysaccharide, bacterial lipoproteins and lipoteichoic acids, flagellin, CpG DNA of bacteria and viruses, double-stranded RNA, and single-stranded viral RNA [60]. The TLR family currently consists of 10 known functional TLRs in humans [61, 62] in which TLR-1 through TLR-9 have been reported in the periodontium, in both health and disease [63]. When Toll-like receptors bind pathogen-associated molecular patterns, a series of intracellular events are initiated, leading to the production of cytokines, chemokines, and antimicrobial peptides (AMPs) [64]. Different Toll-like receptors induce different responses. For example, Toll-like receptors 1, 2, 4, 5, and 6 recognize products that are unique to bacteria and predominate in periodontal tissues, mainly in periodontitis [65] as shown in **Figure 2**.

If gingivitis persists without resolution, bacterial antigens are produced by lymphocytes, macrophages, and dendritic cells. Two different subgroups of lymphocytes, T lymphocytes and B lymphocytes, are released after being exposed with antigens by the innate immune cells. T cells are the effectors of cell-mediated immunity (delayed hypersensitivity), and B lymphocytes carry immunoglobulin

molecules on their surface, which function as antigen receptors [66].

**Figure 2.** *Microbial- and host-associated pathogenesis of periodontal disease.*

#### *Pathogenesis of Gingivitis DOI: http://dx.doi.org/10.5772/intechopen.91614*

*Oral Diseases*

that may induce cell death [40, 41]. GCF originating from the postcapillary venules of the gingival plexus carries blood components like neutrophils, antibodies, and

Saliva, as part of innate immune response, is a key factor in protecting dental enamel, gingiva, and mucosa by flushing microbes and foodstuffs, buffering acids, remineralizing the tooth, providing antimicrobial activity, and permitting selective adhesion of commensal microorganisms to maintain a symbiotic environment in the dental biofilm [43]. The salivary flow rate—high or low—is characteristic of each individual and [44–47] may promote salivary clearance of microbes from the oral cavity. Saliva also contains varying amounts of immunomodulatory interleukin-1β, interleukin-17, and interleukin-23, although it is not known whether they contribute

The recognition of pathogenic microorganisms and the recruitment of effector cells (e.g., neutrophils) and molecules (e.g., the complement system) are central to effective innate immunity. Innate immune responses are orchestrated by a broad range of cytokines, chemokines, and cell surface receptors, and the stimulation of innate immunity leads to a state of inflammation. When microbes penetrate the periodontal tissues, specialized cells of immune system, macrophages and dendritic cells, express a range of pattern recognition receptors (PRRs) which interact with specific molecular structures on microorganisms called microbe-associated molecu-

Biofilms have been defined as "organized microbial communities characterized by a first group of colonizers being irreversibly adhered to a substrate or interphase in a wet media and the rest being embedded in a matrix composed of extracellular polysaccharides produced by the bacteria." The tooth surface provides a non-shedding hard surface where bacteria can adhere and form complex biofilms [8, 49].

The combination of natural host defense mechanisms and oral hygiene practices of individuals helps to have a balanced coexistence of oral microbiota in a healthy oral cavity which can be disturbed by either quantitative (higher bacterial load) or qualitative (growth of pathogenic species) changes in the biofilm leading to early

complement components which help in host defense mechanism [42].

to innate immunity on mucosal surfaces of the oral environment [48].

lar patterns (MAMPs) activating the innate immune responses (**Figure 2**).

**4.2 Bacterial biofilm formation and development of a host response**

*4.1.1 Pathogen recognition and activation of innate response*

**16**

**Figure 2.**

*Microbial- and host-associated pathogenesis of periodontal disease.*

stages of gingivitis [49].

The epithelial attachment of tooth is a highly specialized structure where the junctional epithelial cells strongly attach to the tooth surface by a basal membrane, and hemidesmosomes providing the antibacterial defense mechanism by the high regeneration and desquamation rate and the continuous flow of gingival fluid through the gingival sulcus. The cells of the junctional epithelium with antibacterial proteins like human β-defensin 1 and chemokines along with intercellular adhesion molecule-1 (ICAM-1) and IL-8 help in the migration of PMN toward the gingival sulcus [8].

The protective function of the gingival epithelium is enhanced by keratinization, which helps resist abrasion. The gingival epithelium, as an innate immune barrier, is formed by interconnecting keratinocytes bridged one to another by cell adhesion molecules (CAMs) [50] which include integrins, mediating cell interactions with the extracellular matrix and basement membranes and contributing to cell-cell adhesion [51–53], as well as cadherins, which form tight contacts between cells [54]. The CAMs of the multilayered syncytium are susceptible to digestion by gingipains from *Porphyromonas gingivalis*, which could increase tissue permeability [55–58].

#### **4.3 Innate immune response and gingivitis**

Innate immunity is the first line of defense and the cells responsible for the innate immune response are mainly PMN, macrophages, and dendritic cells. Polymorphonuclear leukocytes (PMNs) are the first and predominant cells of the innate immune system in early gingivitis lesions [13].

The biofilm microbes on the tooth surfaces are recognized by the cells from the innate immunity through certain molecular patterns called pathogen-associated molecular patterns (PAMPs) which include lipopolysaccharide (LPS), peptidoglycans and lipoteichoic acids, N-formylmethionine, and lipoproteins. These molecules are recognized by pattern recognition receptors (PRRs) on the surface of PMNL and macrophages (**Figure 2**).

The two major families of PRRs that have been most extensively studied in the periodontium are the Toll-like receptor (TLRs) and the Nod-like receptors (NLRs) [59]. Toll-like receptors are unique receptors that recognize molecules that are broadly shared by microorganisms but are distinguishable from host molecules and can detect multiple pathogen-associated molecular patterns, including lipopolysaccharide, bacterial lipoproteins and lipoteichoic acids, flagellin, CpG DNA of bacteria and viruses, double-stranded RNA, and single-stranded viral RNA [60].

The TLR family currently consists of 10 known functional TLRs in humans [61, 62] in which TLR-1 through TLR-9 have been reported in the periodontium, in both health and disease [63]. When Toll-like receptors bind pathogen-associated molecular patterns, a series of intracellular events are initiated, leading to the production of cytokines, chemokines, and antimicrobial peptides (AMPs) [64]. Different Toll-like receptors induce different responses. For example, Toll-like receptors 1, 2, 4, 5, and 6 recognize products that are unique to bacteria and predominate in periodontal tissues, mainly in periodontitis [65] as shown in **Figure 2**.

#### **4.4 Activation of adaptive immunity**

If gingivitis persists without resolution, bacterial antigens are produced by lymphocytes, macrophages, and dendritic cells. Two different subgroups of lymphocytes, T lymphocytes and B lymphocytes, are released after being exposed with antigens by the innate immune cells. T cells are the effectors of cell-mediated immunity (delayed hypersensitivity), and B lymphocytes carry immunoglobulin molecules on their surface, which function as antigen receptors [66].

#### *Oral Diseases*

Adaptive immunity provides a more focused defense against infections than innate immune responses, which is slower and dependent on complex interactions between antigen-presenting cells (APCs) and T and B lymphocytes, specifically "cytotoxic T cells" and antibodies. Many histologic studies of periodontal disease [6, 67] have suggested the importance of adaptive immune responses in periodontal pathogenesis by the presence of leukocytes/neutrophils in the early stages of gingivitis and T cells in stable periodontal lesions. The T cells play a major role in maintaining tissue homeostasis against bacterial attack in plaque biofilm [68]. The transition from the established gingivitis lesion to periodontitis is mainly dominated by T and B cells.

#### **5. Host-derived inflammatory mediators**

The molecules participating in the cellular interactions are mainly categorized as proinflammatory and anti-inflammatory, and the balance between these two types of molecules determines the tissue response and the initiation or progression of disease. The key proinflammatory mediators in periodontal disease pathogenesis are as follows.

#### **5.1 Cytokines**

Cytokines are produced by resident cells, such as epithelial cells and fibroblasts, by phagocytes (neutrophils and macrophages) in the acute and early chronic phases of inflammation, and by immune cells (lymphocytes) in established and advanced lesions [69]. Interleukin-1β and interleukin-6 are the main innate cytokines and, together with tumor necrosis factor alpha, are the first to appear in the periodontal disease pathogenesis pathways [70]. Cytokines are effective in very low concentrations and have pleiotropic effects (i.e., multiple biologic activities) on a large number of cell types.

Cytokines are key inflammatory mediators in periodontal disease [71]. They are soluble proteins acting as messengers and binding to specific receptors on target cells to initiate intracellular signaling cascades resulting in cellular changes by altered gene regulation [72, 73]. The genetic regulation leading to the secretion of proinflammatory cytokines from a variety of cells is generally dependent on the activation of nuclear factor kappa-B transcription [74, 75]. The nuclear factor kappa-B-regulated pathways are activated by pathogen-associated molecular patterns, such as lipopolysaccharide, through the Toll-like receptor pathway [75].

#### *5.1.1 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 [71].

IL-1α is an intracellular protein, produced by monocytic, epithelial, osteoblastic cells found in the extracellular environment or in the circulation [76]. Studies have reported elevated IL-1α levels in GCF and gingival tissues in patients with gingivitis and periodontitis [77] and involved in the bone loss that is associated with inflammation [78]. In recent nonhuman primate experiments, the use of a specific IL-1 inhibitor resulted in significant reduction of periodontopathogen-induced attachment loss, bone resorption, and inflammation [79] suggesting that IL-1 inhibitors might be useful in the management of periodontitis.

IL-1β produced by monocytes, macrophages, and neutrophils plays a key role in inflammation and immunity and along with lL-1α induces the synthesis and

**19**

*Pathogenesis of Gingivitis*

*5.1.2 Chemokines*

the gingival sulcus [89, 90].

*5.1.3 Tumor necrosis factor alpha*

*DOI: http://dx.doi.org/10.5772/intechopen.91614*

the bacterial challenge in the periodontal pocket [86].

importance in the pathogenesis of periodontitis.

secretion of other mediators that contribute to the inflammatory changes and tissue damage. IL-1β stimulates the synthesis of PGE2, platelet-activating factor, and nitrous oxide, resulting in vascular changes associated with inflammation [80]. Studies have shown increased concentration of IL-1β in GCF at sites affected by gingivitis [81] and tissue levels of IL-1β correlates with clinical periodontal disease severity [82]. IL-1β increases the expression of ICAM-1 on endothelial cells and stimulates the secretion of the chemokine CXCL8 (IL-8), thereby stimulating and facilitating the infiltration of neutrophils into the affected tissues [83]. Other members of IL family have more roles in the pathogenesis of periodontal disease.

Chemokines are chemotactic cytokines with an important role in the migration of phagocytic cells to the site of infection [84, 85]. Chemokines help in leukocyte recruitment in physiologic and pathologic conditions, which results in the chemotactic migration of neutrophils through the periodontal tissues toward the site of

Chemokines are synthesized by a variety of cells including endothelial, epithelial, and stromal cells, as well as leukocytes [87]. They are divided into two subfamilies: the CC subfamily and the CXC subfamily [88]. The chemokine CXCL8, also known as IL-8, has been found to be localized in the gingival tissues in areas of plaque biofilm accumulation and also in GCF112. Interaction between bacteria and keratinocytes results in the upregulation of IL-8 and ICAM-1 expression in the gingival epithelium, thereby stimulating neutrophil migration into the tissues and

Chemokines target leukocytes of the innate immune system, as well as lymphocytes of the adaptive immune system [91]. Chemokines play important roles in immune responses, repair, inflammation, and regulating osteoclast activity by influencing myeloid cell differentiation into osteoclasts, which may be of particular

TNF-α is a molecularly distinct cytokine and a key inflammatory mediator in periodontal disease that shares many biologic activities with IL-1β [92]. Tumor necrosis factor alpha is a multi-effect cytokine that has many functions, from cell migration to tissue destruction. Tumor necrosis factor alpha impacts cell migration by inducing the upregulation of adhesion molecules and adhesion of neutrophils to the vessel wall, leading to extravasation. It also stimulates the production of chemokines involved in cell migration to infected and inflamed sites [93–96]. The proinflammatory effects of TNF-α include the stimulation of endothelial cells to express selectins that facilitate the leukocyte recruitment, the activation of macrophage IL-1β production, and the induction of PGE2 by macrophages and gingival fibroblasts [97].

**5.2 Lipid mediators of inflammation-prostaglandins and thromboxanes**

and is secreted when stimulated with bacterial lipopolysaccharide [99].

Prostaglandins are derived from the hydrolysis of membrane phospholipids. Prostaglandin E2 (PGE2) and thromboxane B2 are lipid molecules produced by many host cells through the cyclooxygenase pathway, one of the two major paths of arachidonic acid metabolism. Inflamed gingiva synthesizes significantly larger amounts of prostaglandins when incubated with arachidonic acid than in healthy gingiva [98]. Within gingival lesions, prostaglandin E2 is mainly localized to macrophage-like cells

#### *Pathogenesis of Gingivitis DOI: http://dx.doi.org/10.5772/intechopen.91614*

secretion of other mediators that contribute to the inflammatory changes and tissue damage. IL-1β stimulates the synthesis of PGE2, platelet-activating factor, and nitrous oxide, resulting in vascular changes associated with inflammation [80]. Studies have shown increased concentration of IL-1β in GCF at sites affected by gingivitis [81] and tissue levels of IL-1β correlates with clinical periodontal disease severity [82]. IL-1β increases the expression of ICAM-1 on endothelial cells and stimulates the secretion of the chemokine CXCL8 (IL-8), thereby stimulating and facilitating the infiltration of neutrophils into the affected tissues [83]. Other members of IL family have more roles in the pathogenesis of periodontal disease.

## *5.1.2 Chemokines*

*Oral Diseases*

nated by T and B cells.

are as follows.

**5.1 Cytokines**

number of cell types.

*5.1.1 Interleukin-1 family cytokines*

**5. Host-derived inflammatory mediators**

Adaptive immunity provides a more focused defense against infections than innate immune responses, which is slower and dependent on complex interactions between antigen-presenting cells (APCs) and T and B lymphocytes, specifically "cytotoxic T cells" and antibodies. Many histologic studies of periodontal disease [6, 67] have suggested the importance of adaptive immune responses in periodontal pathogenesis by the presence of leukocytes/neutrophils in the early stages of gingivitis and T cells in stable periodontal lesions. The T cells play a major role in maintaining tissue homeostasis against bacterial attack in plaque biofilm [68]. The transition from the established gingivitis lesion to periodontitis is mainly domi-

The molecules participating in the cellular interactions are mainly categorized as proinflammatory and anti-inflammatory, and the balance between these two types of molecules determines the tissue response and the initiation or progression of disease. The key proinflammatory mediators in periodontal disease pathogenesis

Cytokines are produced by resident cells, such as epithelial cells and fibroblasts, by phagocytes (neutrophils and macrophages) in the acute and early chronic phases of inflammation, and by immune cells (lymphocytes) in established and advanced lesions [69]. Interleukin-1β and interleukin-6 are the main innate cytokines and, together with tumor necrosis factor alpha, are the first to appear in the periodontal disease pathogenesis pathways [70]. Cytokines are effective in very low concentrations and have pleiotropic effects (i.e., multiple biologic activities) on a large

Cytokines are key inflammatory mediators in periodontal disease [71]. They are soluble proteins acting as messengers and binding to specific receptors on target cells to initiate intracellular signaling cascades resulting in cellular changes by altered gene regulation [72, 73]. The genetic regulation leading to the secretion of proinflammatory cytokines from a variety of cells is generally dependent on the activation of nuclear factor kappa-B transcription [74, 75]. The nuclear factor kappa-B-regulated pathways are activated by pathogen-associated molecular patterns, such as lipopolysaccharide, through the Toll-like receptor pathway [75].

The IL-1 family of cytokines comprises at least 11 members, including IL-1α,

IL-1β produced by monocytes, macrophages, and neutrophils plays a key role in inflammation and immunity and along with lL-1α induces the synthesis and

IL-1α is an intracellular protein, produced by monocytic, epithelial, osteoblastic cells found in the extracellular environment or in the circulation [76]. Studies have reported elevated IL-1α levels in GCF and gingival tissues in patients with gingivitis and periodontitis [77] and involved in the bone loss that is associated with inflammation [78]. In recent nonhuman primate experiments, the use of a specific IL-1 inhibitor resulted in significant reduction of periodontopathogen-induced attachment loss, bone resorption, and inflammation [79] suggesting that IL-1 inhibitors

IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-33 [71].

might be useful in the management of periodontitis.

**18**

Chemokines are chemotactic cytokines with an important role in the migration of phagocytic cells to the site of infection [84, 85]. Chemokines help in leukocyte recruitment in physiologic and pathologic conditions, which results in the chemotactic migration of neutrophils through the periodontal tissues toward the site of the bacterial challenge in the periodontal pocket [86].

Chemokines are synthesized by a variety of cells including endothelial, epithelial, and stromal cells, as well as leukocytes [87]. They are divided into two subfamilies: the CC subfamily and the CXC subfamily [88]. The chemokine CXCL8, also known as IL-8, has been found to be localized in the gingival tissues in areas of plaque biofilm accumulation and also in GCF112. Interaction between bacteria and keratinocytes results in the upregulation of IL-8 and ICAM-1 expression in the gingival epithelium, thereby stimulating neutrophil migration into the tissues and the gingival sulcus [89, 90].

Chemokines target leukocytes of the innate immune system, as well as lymphocytes of the adaptive immune system [91]. Chemokines play important roles in immune responses, repair, inflammation, and regulating osteoclast activity by influencing myeloid cell differentiation into osteoclasts, which may be of particular importance in the pathogenesis of periodontitis.
