Pattern recognition receptors (PRRs) are a group of cell membrane- and endosome-bound receptors, which can recognize ligands (pathogen-associated molecular patterns or PAMPs) that are broadly shared by pathogens but which are distinct from host molecules [24]. The Toll-like receptors (TLRs) are a key family of PRRs, which play a central role within the innate immune system in the recognition of their ligands or PAMPs. These ligands predominantly include the surface components of bacteria, including lipopolysaccharides (LPS), lipoteichoic acids (LTS), flagellin, peptidoglycans, and lipoproteins as well as nucleic acid ligands from bacterial or viral pathogens. TLR-1 to TLR-6 and TLR-9 expression has been detected in odontoblasts and pulpal fibroblasts, and binding of these PRRs to their respective ligands initiates an acute inflammatory response, leading to activation of cells and release of proinflammatory mediators [16, 25–31]. These molecules include those associated predominantly with the vascular responses of the pulp including histamine, endothelin, serotonin, and neuropeptides and a broad array of cytokines and chemokines with potent cellular signaling properties (Fig. 7.1a–c). Other PRRs include the cytoplasmic NOD-like receptors (NLRs) and retinoic acid-inducible gene (RIG)-like receptors (RLRs) [32], although minimal information is currently available on their involvement in dentin-pulp-mediated inflammation.

An early feature of pulpal inflammation is changes to the vascular flow in the pulp with vasodilation and increases in blood flow. These changes are associated with increased fluid and plasma protein exudation and recruitment of leukocytes. Fluid exudation or edema during acute inflammation classically gives rise to swelling in soft tissues, although, as noted previously, such swelling is constrained in the pulp by the covering hard shell of mineralized dentin. Key molecular mediators of these vascular responses may include histamine, endothelin, neuropeptides, and serotonin. Both in vitro [33, 34] and in vivo [35, 36] studies indicate that histamine can produce vasodilation and reductions in blood flow in the pulp. Endothelin-1, a vasoconstrictor, and its receptors are constitutively expressed in odontoblasts and dental papilla of the developing teeth [37], and its application to pulp causes a decrease in blood flow [38]. A number of neuropeptides have been reported in pulp including substance P, calcitonin gene-related peptide, neurokinin A, neurokinin K, neuropeptide K, neuropeptide Y, somatostatin, and vasoactive intestinal peptide [39], and while these are largely associated with neural structures, some neuropeptides have been reported to be expressed in pulp fibroblasts [40, 41]. A number of these neuropeptides are vasodilators, while others are vasoconstrictors. Their involvement in neurogenic inflammation is complex [39], but these neuropeptides may provide novel therapeutic targets for the control of both pain and inflammation simultaneously [42]. Serotonin, a vasoconstrictor, can stimulate prostaglandin E₂ (PGE₂) [43] and prostacyclin (PGI₂) [44] production as well as increase blood flow in the pulp tissue [45]. PGE₂ and PGI₂ are among a broader family of prostaglandins, which have been implicated in pulpal inflammation [46–48]. Interestingly, histamine synergistically activates COX-2 (one of the cyclooxygenase enzymes involved in prostaglandin generation) expression and PGE₂ production in pulp fibroblasts through a TLR2-mediated process [49].

Several molecular cascade systems, based on plasma proteins, act in parallel to these cell-derived mediators to further initiate and propagate the acute inflammatory response. The complement system is activated by bacteria, with the outcome being the lysis of the bacterial membrane. Early reports have provided rather variable evidence for the contribution of complement activation in the pulp [50–54]; however, these data may reflect the difficulties in detecting the rather transient presence of these proteins during the inflammatory process. Many inflammatory mediators have relatively short half-lives, which in part explains why acute inflammation readily subsides in some tissues once the stimulus has been removed [55]. However, in the pulp, the problems of elimination of the infectious agents and their components ensure that the stimulus will often be ongoing. Complement will likely play a role in leukocyte recruitment (see next section) in the pulp as well as potentially progenitor cell recruitment for subsequent regenerative events [56] (Fig. 7.1a–c). The other molecular cascade systems closely associated with inflammation are the clotting and fibrinolytic systems. Dental pulp has long been recognized to show fibrinolytic activity [57] and the fibrinolytic system may contribute to early wound organization during pulp healing. In the inflamed pulp, gene transcript and protein levels of tissue-type plasminogen activator are increased significantly [58] and are upregulated in the presence of proinflammatory cytokines [58, 59]. As well as allowing plasminogen cleavage to plasmin for fibrin clot lysis, the proteolytic action of plasmin may breakdown C3 facilitating initiation of the complement cascade.

Even at the level of the vascular responses to injurious challenges, the complexity and interrelationships of inflammatory mediator involvement in the pulp are apparent, and our understanding of these events is currently limited.

Recruitment of leukocytes to sites of inflammation is an important aspect of pathogen elimination through phagocytosis and degranulation mechanisms. Increases in vascular permeability facilitate their migration through the endothelial lining, and such extravasation is carefully regulated by the action of molecules involved in their adhesion and transmigration. These molecules include integrins, selectins, endothelial adhesion molecules, and the cell adhesion molecules (intercellular adhesion molecules 1–5, ICAM 1–5; vascular cell adhesion molecule 1, VCAM-1; junctional adhesion molecules, JAMs; platelet endothelial cell adhesion molecule 1, PECAM-1; endothelial cell adhesion molecule, ECAM). While some of these adhesion molecules are constitutively expressed in odontoblasts and other pulp cells in health [60, 61] for the maintenance of tissue architecture, expression of others increases significantly during episodes of inflammation. For instance, weak reactivity for E- and P-selectins in the healthy pulp is strongly upregulated following injury [62, 63].

Recruitment of leukocytes and other cells to sites of inflammation involves attraction along gradients of chemotactic molecules (Fig. 7.1a–c). These chemotactic molecules are diverse in their origins, perhaps reflecting to some extent the lack of specificity of their influences on cell type. Bacterial components are chemotactic to neutrophils in the pulp [64], and components of the dentin matrix released during carious demineralization are chemotactic to both inflammatory cells [65–68] and resident pulp cells [69]. Recognition that the composition of the dentin matrix reflects the expression of a diverse range of molecules by odontoblasts in addition to the well-established structural extracellular matrix components [2, 11] explains why these matrix components have immunomodulatory effects. Certain interleukins are basally expressed by odontoblasts [70] leading to their sequestration within the dentin matrix, and a complex cocktail of pro- and anti-inflammatory molecules have been detected in the dentin matrix [71]. Several inflammatory mediators also show chemotactic properties including complement components C3a and C5a, the arachidonic acid metabolites, and the leukotrienes (especially leukotriene B₄ – LTB₄). Growth factors, cytokines, and chemokines also modulate chemotaxis. The chemotactic effects of some of these molecules influence migration of both pulp progenitor and inflammatory cells [56, 69] highlighting the interplay between inflammatory defense and regenerative events in this tissue following injury (Fig. 7.1a–c).

PAMP recognition by TLRs on odontoblasts and pulpal fibroblasts results in activation of the nuclear factor kappa B (NF-κB) intracellular signaling pathway, which is central to regulation of the molecular inflammatory response in many cell types [16, 25–31]. A range of cytokines and chemokines are produced as a result of activation of NF-κB signaling, and these molecules regulate much of the immune and inflammatory response. These cytokines and chemokines are synthesized by a variety of immune and tissue structural cells in response to infectious and traumatic challenge and have potent cellular signaling properties. Binding of these molecules to specific cell surface receptors further modulates target-cell gene expression and molecular responses via second messenger signaling mechanisms [72, 73]. The actions of these cytokines and chemokines are often synergistic with stimulation of a cascade of release of other related molecules following their initial receptor binding [74]. The immunomodulatory actions of these cytokines and chemokines will impact on both innate and adaptive immune processes, including extravasation, leukocyte recruitment, cell activation and differentiation, and antibody production, as well as regenerative events associated with the wound healing response.

The archetypal proinflammatory regulatory cytokines include interleukin-1α and interleukin-1β (IL-1α,IL-β) and tumor necrosis factor-α (TNF-α), which have been demonstrated to play important roles in pulp’s response to bacterial challenge [66, 67, 75–81]. Dentin matrix dissolution during caries may also stimulate expression of both TNF-α and IL-1β by macrophages [65] emphasizing that environmental sensing and defense mechanisms in the pulp may be broad ranging. The cascades of inflammatory mediator released after early PAMP-PRP interaction are well illustrated by the induction of the proinflammatory cytokine, IL-8 (which is central to neutrophil recruitment and activation), by stimulation with bacterial components or due to IL-1β and TNF-α exposure [70]. Upregulation of IL-8 has been reported in carious human pulp tissue [71, 78, 82], and the constitutive expression of this cytokine in odontoblasts [70] also highlights the complexity of the cellular interrelationships taking place in the defense of the pulp. Both gene and antibody array technologies have allowed demonstration of increased levels of several inflammatory cytokines and S100 transcripts and proteins in carious compared with healthy pulpal tissue [71, 78, 83]. These data are corroborated by other reports demonstrating increased interleukin levels in bacterially challenged pulpal tissue, including increases in IL-4 [84], IL-6 [85], and IL-10 [84]. The release of proinflammatory cytokines within the diseased pulp will have wide-ranging effects generally aimed at reinforcing control of the pathogenic challenge and subsequently resolution of inflammatory processes and stimulation of regenerative events. Among these proinflammatory effects will be the development of chemotactic gradients which promote the recruitment and activation of immune system cells [86, 87] to underpin the innate and adaptive immune responses (Fig. 7.1a–c). These chemotactic mechanisms will operate in tandem with those described previously for leukocyte recruitment to sites of inflammation.

T- and B-lymphocytes, plasma cells, neutrophils, and macrophages are observed to infiltrate the pulp in increasing numbers as carious disease progresses [88, 89] (Fig. 7.1a–c). These cells constitute the effectors of the innate and adaptive immune responses; the latter of which will become increasingly superimposed on the former as caries extends deeply and more extensively into the dentin-pulp and the inflammation becomes more chronic in nature. As caries extends, the immune cell infiltrate in the pulp will also increase and will change from being more focal and localized to a much more extensive presentation. Such changes reflect the transition from more acute to chronic inflammation (Fig. 7.1a–c).

A prime role for the neutrophils and macrophages is that of phagocytosis, especially during the earlier acute phase of inflammation when bacterial pathogens are first encountered. Extravasation of natural killer (NK) cells to sites of inflammation in response to cytokines [90] allows their interaction with immature dendritic cells (DCs), which can lead to reciprocal activation and increased cytokine production by these cells [91]. NK cells likely contribute to further cytokine production during caries including that of IFN-γ [84], which can activate macrophages to stimulate phagocytosis as well as promoting T-cell responses [92]. Tissue-resident DCs are found in the pulp and following PAMP recognition; immature DCs will undergo maturation after which they will likely function in antigen presentation to naïve T cells. T cells have been shown to be present in healthy pulp [93] with CD8+ T cells predominating [88, 89, 94]. An immunosurveillance role is generally assumed for these cells. This contrasts with B cells, which appear to be largely absent from the healthy pulp [89, 93] as also are their plasma cell progeny [51]. However, with establishment of deeper infection within the dentin-pulp, the initial inflammatory cell infiltrate of neutrophils and monocytes intensifies with accumulation of helper T (T _H) cells, cytotoxic T (T _C) cells, regulatory T (T _reg) cells, B cells, and plasma cells as adaptive immune defenses develop [88]. While these immune cells are recruited to the tissue for defense purposes, their ability to achieve clearance of the infection is frequently insufficient, and tissue destruction will often result collaterally. Such tissue destruction may in part be a direct result of the immune cells’ scavenging actions on bacterial pathogens during which release of degradative enzymes and molecules, such as matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) can negatively impact on the host tissue extracellular environment. ROS, which include superoxide anions, hydrogen peroxide, and hydroxyl radicals, can exacerbate tissue injury due to their damaging effects on DNA, proteins, and lipids. Apoptosis can result from cell exposure to ROS through activation of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways [16, 17]. These pathways can be initiated through a variety of cellular stresses, such as cytokine and bacterial LPS exposure, and heat shock [18, 19] highlighting the many opportunities for their activation during infection and inflammation in the pulp. Triggering of these pathways will further stimulate immune cell activity and contribute to an increasing accumulation of inflammatory mediators. It is abundantly clear that the cellular responses initiated during the innate responses associated with acute inflammation and their increasing complexity as adaptive immune responses come into play during chronic inflammation are still poorly defined in the pulp as disease progresses. While many observational studies have reported the presence of various immune cells and their inflammatory mediators in carious pulpal tissue, few functional studies have provided a clear picture of the specific functional activities of these cells and their complex interrelationships. This potentially reflects the individual variation in disease progression in the pulp, which is controlled by diverse factors. Significant challenges still exist to our understanding of the innate and adaptive immune defenses in the pulp, and this constrains clinical approaches to management of pulpal infection and inflammation.

In ideal circumstances, immune defense following injurious challenge to a tissue will lead to elimination of the infecting agent and ultimately provides a conducive environment within which wound healing can occur. Such circumstances are not easily achieved in the dentin-pulp with its noncompliant environment and the exposure of the tooth to the oral cavity with its complex microflora and abundant supply of nutrients. Nevertheless, mechanisms for the regulation of inflammation require both suppression and activation of responses. Identification of several anti-inflammatory and pro-resolving mediators has started to clarify how inflammation may be suppressed after it has achieved its purpose.

The lipoxins are metabolites of arachidonic acid, which negatively regulate the actions of the leukotrienes inhibiting neutrophil chemotaxis and adhesion as well as stimulating apoptotic cell phagocytosis by macrophages and other anti-inflammatory actions [95]. Although there have been no reports of lipoxins in pulp to date, they represent an interesting potential target for control of pulpal inflammation together with the other families of anti-inflammatory mediators described later in this chapter. Three distinct families of anti-inflammatory pro-resolving lipid mediators are now recognized: resolvins, protectins, and maresins [96]. These families target distinct cell populations by interaction with specific receptors and contribute to the overall resolution of inflammation. Resolvins suppress proinflammatory mediator production and regulate neutrophil movement to sites of inflammation. In a rodent model of pulpal infection and inflammation, resolving E1 (RvE1) application led to a decrease in inflammation at 24 and 72 h [97]. Protectins can block T-cell migration and secretion of TNF-α and IFN-γ and promote T-cell apoptosis as well as upregulating the chemokine receptor CCR5 on neutrophils to suppress chemokine signaling. The recently discovered maresins are produced by macrophages and inhibit proinflammatory mediator production by LTA4 hydrolase [98]. The actions of these various specialized pro-resolving anti-inflammatory mediators are only starting to be elucidated, and little information exists on their involvement in pulpal inflammation. Nevertheless, they represent exciting targets for the modulation of inflammatory activity, and the use of analogs may potentially provide novel therapeutic tools for clinical management of inflammation.

The specialized environment of the dentin-pulp can lead to competing pathogenic influences during disease in terms of concomitant release of proinflammatory and pro-reparative/regenerative factors as identified earlier in this chapter. Clearly, a balance in favor of tissue repair is the goal of clinical management of pulpal disease, but this may be unrealistic when relatively high levels of infection and inflammation persist. Current evidence suggests that reparative and regenerative processes ensue only after significant control or resolution of infection and inflammation has occurred [99–101] (Fig. 7.1a–c). Many of the potential cellular signaling mediators present in the post-injury tissue milieu demonstrate pleiotropic effects, which can show dose dependency and contribute to the balance of tissue outcomes. Thus, cytokines and growth factors, such as TNF-α and TGF-β as well as released dentin matrix components, can have detrimental effects on pulpal tissue and induce cell death if present at relatively high concentrations during the infectious and inflammatory processes [66, 68, 102, 103] in contrast to their beneficial effects at lower concentrations. While therapeutic targeting of inflammation may be attractive to change the balance of tissue events, infection control may prove more effective since the inflammatory challenge will continue as long as bacterial involvement persists. However, eradication of bacterial infection of the dental tissues, especially while minimizing host cell damage, has long been a challenge to operative dentistry and endodontics. It is therefore important to better understand the interplay between inflammation and repair/regeneration to guide decision making in the management of dental disease.

It has recently been emphasized that inflammation is an important prerequisite to enable repair and regeneration to subsequently ensue [104]. This may reflect both the need for defense processes to create a conducive environment for repair/regeneration and, also, the pleiotropic effects of some of the proinflammatory mediators, which may impact on repair/regenerative events. A number of proinflammatory mediators can promote degradative events in the oral and dental tissues during their defense responses to pathogenic challenge, for example, in bone resorption in periapical lesions [66, 105]. However, some cytokines, such as TNF-α, can also stimulate pro-regenerative/reparative signaling, including via p38 MAPK pathway activation, leading to odontoblast-like differentiation of dental pulp stem cells with increased dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) expression and tertiary dentinogenesis [106]. The importance of this inflammation-regeneration interplay is further emphasized by the correlation of p38 MAPK signaling with initiation of tertiary dentinogenesis [15]. Such molecular switching is fundamental to the upregulation of odontoblast secretory activity and dentin deposition during the wound healing responses in the pulp. Other proinflammatory mediators, including IL-1β, may also contribute to the interplay of events in the pulp post-injury. IL-1β can stimulate mineralized bone matrix formation by osteoblasts while inhibiting proliferation and differentiation of bone marrow mesenchymal stem cells (BMMSCs) [107]. In the liver, IL-1β can induce the normally quiescent hepatocytes to proliferate, thereby contributing to regeneration of this organ [107]. In the context of the dentin-pulp, proinflammatory cytokines may stimulate wound healing in surviving odontoblasts at earlier stages of disease when the pathogenic challenge is relatively less intense. However, during chronic disease, these cytokines may suppress odontoblast-like cell differentiation from stem/progenitor cells until infection is controlled and a more conducive tissue environment for wound healing prevails (Fig. 7.1a–c).

Other inflammatory mediators may also influence post-injury events in the pulp. While immune cell-derived ROS can contribute to tissue damage, at relatively low levels, these molecules can promote stem/progenitor cell differentiation and mineralization [108]. Clearly, there is a complex interplay occurring between the various molecules mediating events in the pulp post-injury, and the relative concentrations of these molecules may be key to the cellular signaling outcomes. Thus, at relatively low concentrations, these molecules may stimulate regenerative/reparative events, including cellular recruitment, differentiation, and matrix secretion. At higher concentrations, however, such events may be impeded through signaling inhibition and tissue degradative processes (Fig. 7.1a–c). Elucidation of these inflammation-regeneration interplay relationships is complicated by the various origins of these mediator molecules. Contributory sources will be tissue resident and immune cells as well as dentin matrix sequestrated pools released during carious dissolution. The relative contributions from these various sources will fluctuate with both the rate of disease progression (and hence, carious matrix dissolution) and the extent of cellular signaling taking place in the tissue (Fig. 7.1a–c). Some of the cytokine receptors responsible for triggering of cellular responses are common to both the immune/inflammatory and stem cells, which may explain the pleiotropic effects of these cytokines. For example, C-X-C chemokine receptor 4 (CXCR4) is expressed on lymphocytes and granulocytes and is also involved in stem cell recruitment [109, 110]. Both stromal cell-derived factor-1 (SDF-1)/CXCL12 and its receptor are expressed in pulp and are upregulated during disease [111, 112]. The sharing of common cytokine receptors between immune and stem cells probably reflects evolutionary conservation of cellular signaling processes. Recruitment of both cell types is required post-injury, and the extent of injury or infection may be the prime determinant of the level of involvement of these different cell types [113]. Further regulatory control of tissue events in the inflammatory milieu may also be triggered through modulation of stem cell cytokine receptor expression. For instance, increased cytokine levels can modulate the surface expression of CXCR4 on stem cells [109]. Such modulation could result in the suppression of regenerative/reparative responses during active inflammation.

In addition, mesenchymal stem cells (MSCs) can display immunomodulatory effects in tissues further demonstrating the dynamic interplay between inflammation and regeneration [114]. Dampening of excessive inflammatory responses by MSCs through modulation of immune cells will likely involve a number of mechanisms. One such process has recently been identified in which MSCs have been demonstrated to inhibit the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in monocytes/macrophages by decreasing ROS generation [115]. Although the scope of the immunomodulatory properties of dental pulp stem cells is still being explored [20–22