Overlapping Protective and Destructive Regulatory Pathways in Apical Periodontitis

Review Article

Overlapping Protective and Destructive Regulatory Pathwaysin Apical PeriodontitisIldik�o J. M�arton, MD, LDS, PhD, DSc,* and Csongor Kiss, MD, PhD, DSc†

Abstract
Introduction: Protective and destructive immunoreac-tions take place simultaneously in apical periodontitis.However, the same reactions defending the periapicalarea from infection-derived damage may also result inhost tissue injury. Methods: The inflammatory reactionof the periapical tissues is self-limited. Regeneration ofthe injured tooth-supporting structures may follow elim-ination of the causative microbial irritation. Results:Recent experimental and clinical observations haveidentified important interplay between positive andnegative regulatory pathways. A network of stimulatoryand inhibitory feedback loops may influence the inten-sity of the defense and inflammatory responses andthe balance between bone resorption and regeneration,resulting in lesion expansion or healing of apical peri-odontitis. Conclusions: We critically discuss researchdata on regulatory mechanisms that control the activityof host effector cells and signaling molecules during in-teractions with pathogenic microbes. (J Endod2014;40:155–163)
Key WordsApical periodontitis, bone resorption, cytokines, immunity,inflammation, stem cells

From the *Department of Restorative Dentistry, Faculty ofDentistry and †Department of Pediatric Hematology-Oncology,Institute of Pediatrics, Faculty of Medicine, Medical and HealthScience Center, University of Debrecen, Debrecen, Hungary.

Address requests for reprints to Dr Ildik�o J. M�arton, Medicaland Health Center, University of Debrecen, 98 Nagyerdei Krt,Debrecen H-4032, Hungary. E-mail address: [email protected]/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.10.036

JOE — Volume 40, Number 2, February 2014

Apical periodontitis, an inflammatory response within the periapical tissues, mostfrequently results from polymicrobial infection of pulpal origin. The disease may

take an acute or chronic course. Chronic apical periodontitis may present as dentalgranuloma or periradicular cyst (1). Based on clinical and experimental reportsfrom the 80s and 90s, we have proposed that protective and destructive immunoreac-tions take place simultaneously in chronic periapical lesions (2). However, ‘‘destruc-tive’’ and ‘‘protective’’ terms need to be carefully defined because the same reactionsresponsible for host defense by the elimination of invading microorganisms may alsoresult in tissue injury. Moreover, tissue injury can be prevented by the inhibition of anti-microbial immunoinflammatory reactions, albeit at the price of overwhelming infection(3, 4). Here, we review the results of recent animal and human studies and laboratoryexperiments that contribute to a better understanding of the periapical inflammatoryprocesses. Furthermore, we analyze regulatory mechanisms that control the activityof effector cells andmolecules. These mechanisms can influence the fine-tuned balancebetween protection from apical infection, lesion progression, and repair. Table 1 sum-marizes the major subclasses of immune cells playing an important role in apical peri-odontitis, their major actions, and examples of major released mediators.

Regulation of the Initial Inflammatory Cell AssemblyProliferative activity is restricted to epithelial cells (ECs) in periapical lesions

(5, 6). A robust influx of leukocytes is initiated by the interaction betweenmicroorganisms of the infected root canal and the periodontal ligament (PDL)(7–10). PDL cells constitutively express low levels of adhesion molecules andchemokines. They provide a weak stimulus for recruiting and activating leukocytes,thereby maintaining the homeostasis of healthy periapical tissues (11–14).Pathogenic bacteria and bacterial molecules released from the infected root canalstimulate PDL cells to up-regulate a broad set of inflammatory mediators. These includechemoattractant molecules, which stimulate the influx of inflammatory cells to thelesion site (7, 9, 15, 16). Human PDL fibroblasts (PDLFs) express toll-like receptor(TLR)-related molecules (ie, TLR2, TLR4, MD-2, and MyD88) and CD14. Using theCD14/TLR pathway, PDLFs challenged with lipopolysaccharide (LPS) and Staphylo-coccus epidermidis peptidoglycan initiate an interleukin (IL)-8/CXCL8 response, thekey signal for polymorphonuclear (PMN) leukocyte chemoattraction (17) (Fig. 1).As shown in cultured cells, TLR receptors may stimulate inflammation independentlyof the CD14 molecule. The synthetic peptidoglycan analog muramyl dipeptide inducesa positive regulatory loop via nucleotide-binding oligomerization domain-like receptorsin apical periodontitis (17–19). Porphyromonas gingivalis and LPS extracted from P.gingivalis and from Escherichia coli increase gene expression of both pro- and anti-inflammatory cytokines in human PDLF cell cultures. The former ones are representedby tumor necrosis factor alpha (TNF-a), IL-6, monocyte chemoattractant protein(MCP)-1/CCL2, the chemoattractant ‘‘regulated upon activation, normal T-cell ex-pressed and secreted’’ (RANTES)/CCL5, and stromal-derived factor 1 and the latterones by transforming growth factor-beta (TGF-b). The pattern and intensity of cytokinegene expression by PDLFs shows marked variation both between different individualsand depending on the source of the stimulatory agent (20–22). In lesion-derived hu-man PDLF extracts, 54% of the genes investigated with an inflammatory gene array weresignificantly up-regulated compared with healthy PDLF extracts (23).

Herpesviruses including Epstein-Barr virus, cytomegalovirus, and human herpes-virus 6, but not human herpesvirus 8, contribute to the etiopathogenesis of human apical

Protective and Destructive Regulatory Pathways 155

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TABLE 1. Major Subclasses of Immune Cells and Their Major Actions

Subclasses of immune cells Major actions Major released mediators

Antigen presenting cells (APC) Uptake and processing of antigens anddisplaying them to T cells

Depending on the subtype of APC (seebelow)

B cells/lymphocytes Formation of antigen-specific antibodies;performing as APCs

Fully activated B cells (plasma B cells/plasma cells) secrete antibodies

DCs Function as professional APCs, innaterecognition of microbes by TLR,regulate other immune cells includingT and B cells

mDCs produce TNF-a and cytokines of theIL-1 and IL-12 families; pDCs producetype I interferons

LCs mDCs of the mucosa and skin Similar to other mDCsMacrophages (together with monocytes,

macrophages are called MNPs)Develop from monocytes; function as

professional phagocytes, and APCs;release toxic compounds that destroymicrobes and innocent bystander hostcells; regulate immune cells

Cytokines: IFN-b, IFN-g, IL-1a, IL-1b, IL-6,IL-10, IL-15, IL-18, migration inhibitoryfactor, TGF-b TNF-a; chemokines:CCL-2CCL-3,CCL-4, CCL-5, CCL-22, IL-8,macrophage inflammatory proteins;reactive oxygen and nitrogenintermediates; eicosanoids; proteases

Mast cells Anaphylactic type of allergic reactions;nonspecific antimicrobial defense;wound healing; immunoregulation

Histamine, serotonine; eicosanoids;cytokines: IL-1b, IL-3, IL-4, IL-5, IL-13,GM-CSF, SCF; chemokines: CCL-2, CCL-5,eotaxins

MOs Phagocytosis, antigen presentation, giverise to mDCs and macrophages

Similar to mDCs and macrophages

NK cells Cytotoxic lymphocytes of innate immunity,contribute to self- tolerance andimmune memory

Cytotoxins: perforin, cytotoxins: perforin,chemokines CCL3, CCL4, and CCL5;cytokines IFN-g, GM-CSF, and TNF-a

NKT cells CD1d-restricted T cells; recognize lipidsand glycolipids; rapid release of solubleregulatory mediators

Cytotoxins as NK cells; IL-2, IL-4, IL-13,IL-17, IL-21, IFN-g, GM-CSF, TNF-a

PMNs Of the 3 types of PMNs (basophilic,eosinophilic, and neutrophilic),neutrophils play a major role in apicalperiodontitis: provide first line ofdefense against pathogens byphagocytosis; attract and stimulatefurther PMNs, MOs, and macrophages;destroy periapical tissue components

Reactive oxygen and nitrogenintermediates; proteins withantimicrobial activities: acid hidrolases,defensins, lactoferrin, lysozyme;proteases destroying microbes and hosttissues; eicosanoids; cytokines andchemokines: IL-1b, TNF-a, IL-8, andCXCL-10

T cells/lymphocytes Participate in cell-mediated immunereactions as effectors and regulatorycells

Depending on the subtype of T cells

Cytotoxic T cells (CD8-positive T cells) MHC class I–restricted cells, destruction ofinfected cells

Cytotoxins: NK cells; cytokines: IFN-g, IL-2,GM-CSF; TNF-a; chemokines: CCL-3,CCL-4, CCL-5

Memory T cells (CD45RO-positive cells) Rapid expansion to effector T cells uponantigen re-exposure

IFN-g, IL-4, IL-17

Helper T cells (Th) (CD4-positive T cells) MHC class I–restricted cells;promotematuration of B cells, activation of Tcellsand macrophages; differentiate intoseveral subtypes, which producestimulatory and inhibitory cytokines

Activated Th cells: IL-2; subset of activatedTh cells: IL-17; Th1 cytokines: IFN-g,TGF-b; Th2 cytokines: IL-4, IL-5, IL-6,IL-10, IL-13; Th17 cytokines: IL-1, IL-6,IL-17, TNF-a

Regulatory T cells (Treg) (CD4+/CD25hi/Foxp3+ cells)

Suppress activation of the immune systemby secreting regulatory cytokines and bycell-to-cell contacts

TGF-b and IL-10

APCs, antigen-presenting cells; DCs, dendritic cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-b, interferon beta; IFN-g, interferon gamma; IL, interleukin; LCs, Langehans cells;

MDCs, myeloid dendritic cells; MNPs, mononuclear phagocytes; MOs, monocytes; NK, natural killer; NKT, natural killer T; pDCs, plasmacytoid dendritic cells; PMN, polymorphonuclear leukocyte;

TLR, Toll-like receptor; TNF-a, tumor necrosis factor alpha.

Review Article

periodontitis by stimulating an increased production of early inflamma-tory cytokines and chemokines (24–27). Synergistic interactionsbetween herpesviruses and endodontopathogenic bacteria mayexacerbate the severity of the inflammatory response within theperiradicular area (28–30). An aggressive type of pathologic pictureand a symptomatic manifestation often characterize herpesvirus-productive periapical infection.

In vivo and in vitro studies showed that the production of chemo-attractant molecules can be induced by irritation from dental materials,implants, and orthodontic tooth movement (31–33). Dentin proteins

156 M�arton and Kiss

can augment the migration of neutrophil leukocytes by the inductionof KC/CXCL1 and MIP-2/CXCL2 in mice (34). Sensory neuropeptidesubstance P, which is released during painful stimuli, has been shownto induce the expression of MIP-3a/CCL20 in immortalized PDLF cells.Concomitantly, substance SP stimulated the expression of the stressenzyme heme oxygenase 1 as a down-regulatory signal (35).

The first wave of chemokines released from pulpal and PDL cellsreach the blood vessels, increasing their permeability and selectively at-tracting white blood cells to the site of inflammation (8, 16, 36).Chemokines bind to the molecules of the extracellular matrix within


Figure 1. A schematic representation of the regulation of the initial inflammatory cell responses in the periapical area. The interaction of LPS and TLRs expressedon PDLFs initiates the production of proinflammatory cytokines and chemokines IL-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, pro-IL-1b, IL-6, stromal-derived factor 1,and TNF-a as well as the immunoregulatory compounds TGF-b, IL-1Ra, and sTNF-aR. Proinflammatory mediators induce vasodilation and attract PMNs and MNPsto the periapical area. Invading bacteria, their byproducts, and the first wave of inflammatory mediators activate phagocytes. These cells produce a broad set ofsoluble mediators, which destroy pathogenic microbes, injure tooth-supporting tissues, and attract further cells of the innate and adaptive immune system. Incontrast, immunoregulatory compounds attenuate the intensity of inflammatory reactions and host tissue destruction (see text for references).

Review Article

the lesion providing a permanent attraction stimulus for migrating cells(37). Cells of the adaptive immune system migrate with a lag period of2–3 days after the initial infiltration of the periradicular area by neutro-phils and macrophages in experimental animals with exposed root ca-nals (38). In parallel with lesion size expansion after days 21–56 ofpulp exposure, protective and destructive mechanisms reach a dynamicequilibrium (39). Analysis of the expression profile of a 30,000 genedata set in a rodent model of apical periodontitis revealed a networkof interrelated regulatory loops. The differential expression of 2 mono-cyte-/macrophage-derived cytokines, IL-1a and IL-1b, was describedduring the inflammatory and healing phases. This observation suggeststhat IL-1b may be involved in reactions contributing to cellular diversi-fication of the early lesion and T-lymphocyte–dependent antibody pro-duction in later phases, whereas IL-1a may promote healing (40).Similarly, in human primary endodontic infections with apical peri-odontitis, individual cytokines were shown to correlate with lesionscharacterized by different clinical and radiographic appearances. IL-1b and TNF-a correlated with the presence of exudate during thecourse of root canal treatment. IL-6 exhibited a negative correlationwith lesion size (41).

Participation of Cellular Components inPeriapical Protective and Destructive Reactions

Mononuclear phagocytes (MNPs) are among the initially recruitedcells in early apical periodontitis. These cells participate both in protec-tive and destructive reactions. MNPs directly eliminate microbes


invading from the infected root canal and initiate antigen-specific re-sponses by processing and presenting antigens to T lymphocytes.They contribute to the establishment of the lesion by destroying healthyperiapical tissues (4, 42–44). Exudative macrophages express a varietyof chemokine receptors. Multiple chemokines, including MCP-1/CCL2,MIP-1a/CCL3, and RANTES/CCL5, and IL-8/CXCL8, the main neutrophilchemoattractant, may mediate MNP migration to the periapical lesion(36). The expression of these ligand-receptor pairs at the RNA and pro-tein levels has been reported in experimental and human periapical in-flammatory lesions (37, 40, 45–47). Stimulated MNPs produce a broadset of soluble molecules that attract further cells to the lesion and up- ordown-regulate their activities (Fig. 1). Advanced immunophenotypingmethods and ex vivo functional assays have revealed important detailsof the cross-talk between antigen-presenting cells (APCs) and T lym-phocytes (48, 49). In clinical samples, CD14+/HLA-DR+ activatedmacrophages made up about 17% of the mononuclear cells (MNCs)isolated from periapical lesions (50). Langerhans-type dendritic cells(DCs) made up 12%–15% of the total DC population and were foundto be associated with CD3+ T-lymphocyte infiltration and EC prolifera-tion (44, 51, 52). Interestingly, the predominant subpopulation oflesion-associated APCs was represented by HLA-DR+/CD19+ B lym-phocytes (44, 50).

Investigations of experimental rodent models and human samplesin the 90s suggested that T-helper type 1 (Th1) lymphocytes character-ized early forms of apical periodontitis. This suggests a role for Th1 cellsin the initiation and expansion of lesions. The abundance of B lympho-cytes, plasma cells, CD8+ cytotoxic/suppressor T cells, and Th2 cells in


Review Article
late periapical granulation tissues suggests that these cells participate inlesion stabilization and healing (53–56). However, recent clinical andexperimental findings indicate that the inhibition of helper T-lymphocyte function can contribute both to the attenuation andexacerbation of local inflammation and tissue destruction in apicalperiodontitis (57–59). The secretion of 2 anti-inflammatory cytokines,TGF-b and IL-10, showed a positive correlation with the ratio of CD8+ Tlymphocytes in large-sized periapical lesions and in lesions predomi-nated by B lymphocytes and plasma cells (‘‘B-type’’ lesions). These ob-servations suggest the importance of immunosuppressive reactions inthe late phase of lesion development in human apical periodontitis(44, 49). The overexpression of Th1-type (CCR1, CCR5, and CXCR3)and Th2-type (CCR2 and CCR3) chemokine receptors in individual hu-man periapical lesions compared with healthy tissue indicates theimportance of simultaneous action of both types of responses in diseasepathogenesis (45, 47). The frequency of Th1 cells consistentlyexceeded that of Th2 cells in every lesion type. Th1-type cytokines(IL-2, interferon gamma [IFN-g], and IL-12) were identified in100% of lesions, whereas Th2-type cytokines (IL-4 and IL-5) were de-tected only in a restricted number of cell culture supernatants from hu-man apical periodontitis lesions (43, 44). Mast cells, consistentlypresent in periapical granulation tissue, may contribute to Th2-type po-larization (60, 61). Two recently described T-helper cellsubpopulations, T-helper cell type 17 (Th17) and regulatory Tlymphocytes (CD4+/CD25hi/Foxp3+ ‘‘Treg’’ cells), were shown tocontribute to the cellular networking in advanced apical periodontitislesions (38, 46, 62, 63).
ECs are present in approximately 50% of periapical lesions. Their3-dimensional strands may merge to form true periradicular cysts andperiapical pocket or ‘‘bay’’ cysts. ECs have been suggested to perpetuatethe periapical inflammatory process (5, 64, 65). Epithelializedperiapical lesions are characterized by the production of high levelsof IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the presence of ECs was correlated with clinical symptomsin patients with chronic apical periodontitis (9, 65, 66). Naturalkiller cells (CD56/57+/CD3-) and natural killer T cells (CD56/57+/CD3+) make up approximately 1% of the granulation tissue. Theyhave been suggested to promote proapoptotic pathways for ECswithin the lesion. In cooperation with plasmacytoid DCs, theyproduce type I interferons with antiproliferatory and antiviralactivities (43, 67, 68).

Lesion expansion is associated with angioneogenesis in chronicinflammatory diseases. Vascular endothelial growth factors (VEGF)and their receptors (VEGF-R) play a critical role in angiogenesis. Theexpression of VEGFs and VEGF-Rs, in particular VEGF-A, -C, and –D,as well as VEGFR-2 and -3, has been shown immunohistochemicallyin human apical periodontitis of chronic nature. In addition to locali-zation on blood vessels, VEGFs and VGEF-Rs were also identified on neu-trophils, macrophages, and T and B lymphocytes. Up-regulateddownstream mediators of VEGF-mediated angiogenic activity wereshown in these samples by quantitative real-time polymerase chain re-action and compared with healthy PDL samples (69, 70).

Network of Regulatory Loopsin Chronic Apical Periodontitis

Results of immunophenotyping, gene expression analysis, andfunctional investigations indicate a key regulatory role for monocytesand DCs in the induction of antigen-specific T-cell responses. Moreover,these cells are essential for the polarization of the T-helper immune re-sponses toward Th1, Th2, Th17, and Treg pathways in advanced apicalperiodontitis. DCs andmacrophages differentially produce Th1 and Th2


cytokines. The former cells represent the major source of IL-12 and ofIL-23. DCs, cocultured in vitro with allogeneic T cells, secrete highlevels of IFN-g and stimulate the proliferation of CD4+ lymphocytes.A close association between CD83+ DCs and CD3+ T cells in humansamples has been observed with confocal laser scanning microscopy(42). In lymphocyte-rich areas, up-regulated expression of HLA-DRa-chain, CD83, and CD86 in DCs and CD28 in T lymphocytes, respec-tively, was observed (48). These observations suggest that DCs presentin human periapical lesions are able to process microbial antigens, acti-vate na€ıve T cells, support both Th1 and Th17 reactions, and are essen-tial for granuloma formation (71–73) (Fig. 2).

Macrophages may mediate protective and destructive stimuli at thesame time. Macrophages serve as APCs for effector T cells and producematuration factors for DCs, proinflammatory cytokines, arachidonicacid derivatives, and reactive oxygen intermediates stimulating boneresorption. Monocytes down-regulate the production of IFN-g in DC/CD4+ cell cocultures. Macrophages produce IL-10 in cooperationwith Th2 and Treg cells and IL-27 in cooperation with Treg cells(71) (Fig. 2). These 2 cytokines were shown to exert potent immuno-regulatory activities, which restrict untoward proinflammatory re-sponses and bone resorption in human periapical lesions (49, 72, 73).

IL-1b, produced by activated macrophages, is a primordial cyto-kine of the inflammatory phase in experimental periodontitis, mediatinglesion expansion (40, 74, 75). High levels of IL-1b, caused bypolymorphic alleles of the encoding gene alone or in combinationwith polymorphisms of genes encoding for IL-6 and TNF-a, are associ-ated with symptomatic periapical abscesses and root canal treatmentfailure (76, 77). Negative feedback mechanisms prevent IL-1b–induced severe destruction of periodontal tissues. In human samples,IL-1b and IL-6 induce suppression of cytokine signaling-3 proteinexpression, thereby diminishing bone resorption through achemokine-dependent mechanism. Moreover, IL-1b directly inhibitshuman osteoclast precursors (78, 79).

Th17 cells are present in all phases of induced rat periapical lesions,and their numbers increase during lesion expansion (63). IL-17–positivecells have been shown in human dental granulomas and radicular cystswhere they correlated with the presence of sinus tracts. The number ofTh17 cells was higher in periapical lesions than in control samples(46). IL-17 was invariably detected in lesion-derived MNC culture super-natants, and its level was significantly higher in symptomatic lesions thanin asymptomatic ones. The ability of IL-17 to induce neutrophil attractionand the activation of the soluble mediators GM-CSF, IL-1, IL-6, IL-8/CXCL8, and GROa/CXCL1 were shown to correlate positively with the ratioof neutrophils in periapical lesions (44, 80). Interestingly, completeabrogation of IL-17 responses, as shown in IL-17 receptor (IL-17RA)-deficient mice, resulted in enhanced periodontal bone destructioninduced by P. gingivalis. This indicates a nonredundant role for IL-17in mediating host defense via neutrophil mobilization (81).

TGF-b is a potent down-regulatory factor for IL-17–induced in-flammatory responses although the two cytokines do not exert mutualinhibitory effects (82). The major source of TGF-b is CD4+/CD25hi/Foxp3+ Treg cells, making up 30%–50% of all activated T-helper lym-phocytes (CD4+/CD25+ cells) in human periapical lesions (62, 83,84). The antiproliferative activity of lesion-derived MNCs on autologousand allogeneic peripheral blood-derived MNCs depended on the pres-ence of IL-10 and Tregs within the cell suspension (62). Clinical andexperimental observations suggest that the balance between Th17and Treg cells is critical to the outcomes of periapical infection in termsof ‘‘destruction’’ (lesion expansion with or without symptoms) or ‘‘pro-tection’’ (healing). The Th17/Treg ratio may influence the intensity ofthe inflammatory response, the efficacy of local and systemic antimicro-bial defense reactions, and the activity of processes resulting in bone


Figure 2. The balance of Th17 and Treg cells regulates the efficacy of antimicrobial defense reactions, the intensity of destructive inflammatory responses, and therepair processes. Th17 lymphocytes produce the master proinflammatory cytokine IL-17, which, by inducing the secretion of GM-CSF, IL-1, IL-6, IL-8/CXCL8, andGROa/CXCL1 and the expression of RANKL, results in the recruitment of PMNs and MNPs and enhances osteoclastogenesis. The most potent inducers of Th17differentiation and IL-17 expression are IL-1b and IL-23, which are produced mainly by DCs activated by bacterial molecules and proinflammatory cytokines.CD4+/CD25hi/Foxp3+ Treg cells express TGF-b and IL-10. The latter cytokine, together with IL-27 produced by Treg cells in cooperation with monocyte-derived APCs, suppresses IL-8/CXCL8 release by inhibiting Th17 lymphocytes. TGF-b is considered as the major ‘‘protective’’ factor in apical periodontitis. Itdown-regulates IL-17–induced inflammatory responses. However, in the presence of monocyte-derived APCs, IL-2 and IL-15 Treg cells can transdifferentiateinto Th17 lymphocytes, probably through a small subset of double-positive IL-17+/Foxp3+ cells (see text for references).

Review Article

resorption or regeneration. Treg and Th17 cells, characterized by areciprocally interrelated differentiation program, may not only inhibitbut also support the differentiation and expansion of one and another.TGF-b, considered as one of the major ‘‘protective’’ factors in apicalperiodontitis, may drive Th17 polarization of na€ıve CD4 T-lymphocytesin the presence of IL-1b or IL-6 (85, 86). Moreover, human Treg cellscan transdifferentiate into IL-17–producing cells in the presence ofAPCs and exogenous IL-2 and IL-15 (87). The plasticity of the T-celllineage may depend in part on the presence of a small subset of doublepositive T-helper lymphocytes coexpressing ‘‘lineage-restricted mole-cules’’ (eg, IL-17 and Foxp3). These partially differentiated T cellsmay retain their capability to become IL-17–producing cells (Fig. 2).Such cells have been observed in human inflammatory lesions,including apical periodontitis. They have been suggested to play atwin role in antimicrobial defense and restricting the intensity of inflam-matory responses (44, 88).

Molecular Regulation of Effector MechanismsThe first step in lesion development is the destruction of extracel-

lular matrix by serine proteases and matrix metalloproteinases (MMPs)secreted from the lysosomal granules of phagocytes. In experimental ratperiradicular lesions, the number of elastase-expressing neutrophilgranulocytes andmacrophages increased up to 28 days after pulp expo-sure. Cells expressing the secretory leukocyte protease inhibitoremerged after day 28 in parallel with the start of lesion shrinkage(89, 90). PDLFs represent additional sources of MMPs, which areusually secreted as latent proenzymes and become activated by otherhuman endopeptidases, bacterial proteases, and reactive oxygenintermediates. MMP synthesis and release are tightly controlled by


the local cytokine milieu, endotoxins, and cell-to-cell contacts(91–93). The presence of a broad set of MMPs at the messengerRNA and protein levels has been widely observed in inflamed but nothealthy periradicular tissues. This applies to both patients andexperimental animals (10, 39, 94–98). Significantly higher numbersof gram-negative bacteria and elevated MMP-9 expression have beenobserved in patients with symptomatic periapical lesions whencompared with asymptomatic patients (99). The kinetics of MMP up-and down-regulation in experimental apical periodontitis models isassociated with lesion expansion and stabilization and is in accordwith a dominant role for IL-1 in early expanding periapical lesionsand the immunoregulatory actions of TGF-b during later stages (39,91, 100). Polymorphisms of MMP2 and MMP3 genes, encoding forMMP-2 and -3, were reported to be associated with an increased riskof developing periapical lesions as well as predicting healing responsesin patients with advanced caries (101).

Effector molecules produced by phagocytes may be key therapeu-tic targets in periapical disease. In addition to the well-established bene-ficial effects of Ca(OH)2 for pulp capping in humans, this root canaldressing was shown to decrease the number of MMP-positive cells inexperimental apical periodontitis (2, 10). In contrast, systemicantioxidant therapy failed to prevent lesion development or todecrease lesion size of induced apical periodontitis in a rat model oftype 2 diabetes (102).

The equilibriumbetween bone formation and resorption in the peri-apical region relies on the well-recognized interactions between osteo-blasts and osteoclasts involving the stimulatory action of receptoractivator of nuclear factor kappa-B (RANK)-RANK ligand (RANKL). Themajor negative regulator of the RANKL-RANK pathway is the soluble decoyreceptor osteoprotegerin (OPG) (103) (Fig. 3). In addition to


Figure 3. The equilibrium between bone formation and resorption is regulated by the interaction between osteoblasts and osteoclasts and their precursors.Membrane-bound RANKL expressed in osteoblasts stimulates the differentiation of osteoclast precursors and the bone-resorbing activity of mature osteoclaststhrough interaction with its receptor RANK. The soluble form of RANK, OPG, inhibits RANKL-RANK interaction. RANKL-RANK signaling activates nuclear factor-kB. Osteoclasts are descendants of the bone marrow–derived monocyte/macrophage lineage and are characterized by the expression of tartarate-resistant acidphosphatase (TRAP). The osteoclast releases hydrogen ions (H+) through the action of carbonic anhydrase, resulting in a low pH environment in the bone-resorbing lacuna. Bicarbonate (HCO3

�)-chloride (Cl�) ion exchange restores the intracellular pH of this cell. The expression of RANKL and OPG is reciprocallyregulated by IL-1, IL-6, IL-11, IL-17, M-CSF, GM-CSF, and TNF-a, which induce bone resorption. sRANKL is an additional stimulator of bone resorption. LPS maycontribute to the differentiation of osteoclast precursors. In contrast, IFN-g and IL-10 decrease the RANKL-OPG expression ratio and attenuate bone resorption. Inthe bone formation process, osteoblasts differentiate into osteocytes.

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osteoblasts, monocytes, DCs, fibroblasts, and endothelial and epithelialcells, PMNs and activated T lymphocytes express RANKL in apical peri-odontitis lesions from patients and experimental animals. The widespreadmanifestation of RANKL may explain the characteristic shift toward bonedestruction (104–106). Expression levels of both RANKL and OPG inhuman dental granuloma samples were higher than in healthy PDLcells, indicating an elevated bone turnover in apical periodontitisversus noninflamed periapical tissues. The ratios of RANKL and OPGexpression exhibited a heterogeneous pattern suggesting that somelesions were in the stage of active bone resorption, whereas otherswere at the stable chronic or healing stages (105, 107). The numbersof osteoclasts and the RANKL:OPG expression ratios show parallelincreases in expanding experimental periapical lesions (79, 108, 109).TNF-a, IL-1b, IL-6, and IL-17 reciprocally regulate the expression ofRANKL and OPG favoring RANKL expression (110, 111). TNF-a, IL-1a,and macrophage migration inhibitory factor induce further osteoclaststimulating molecules such as IL-6, IL-11, IL-17, M-CSF, RANKL, andprostaglandin E2 in human dental pulp cells and induced rat periapicallesions (112, 113). Periodontopathogenic bacteria and their constituentsinduce RANKL andOPG expression in PDLFs and dental pulp cells directlyor by up-regulating osteolytic cytokines, such as TNF-a and IL-1b(114–116) (Fig. 3).

In contrast, some other cytokines, including IFN-g, decrease theRANKL:OPG expression ratio, thereby attenuating bone resorption(110). The osteoprotective role of IFN-g, together with IL-10, intercel-lular adhesion molecule-1 (ICAM-1), and CCR5, was confirmed inmice with genetic ablations of the genes encoding for these factors(117) (Fig. 3). Experimental observations indicate that direct cross-talk between IFN-g and the RANKL-RANK signaling pathway and the


inhibition of Th17 polarization by IFN-gmay contribute to bone protec-tion (118, 119). Other bone-protective mechanisms do not directlyinvolve the RANKL-RANK signaling pathway. In experimental rat apicalperiodontitis, the expression of the non–DNA-binding intracellular coac-tivator was inversely related to osteoblast activity and lesion progression(120). In a similar experimental model, the cholesterol-lowering agentsimvastatin suppressed bone resorption and decreased the expression ofthe angiogenic cysteine-rich 61 protein. This was carried out by enhance-ment of the transcription factor Forkhead/winged helix box protein O3a(FoxO3a). These in vivo results have been corroborated in a murineosteoblast cell line (121).

Regulation of Repair MechanismsTissue destruction and bone loss is self-limiting in most cases of

apical periodontitis. Induced apical periodontitis in rats showed adecrease in the number of infiltrating osteoclasts accompanied bylesion size stabilization after 21 days after pulp exposure. Thus, a spon-taneously established new equilibrium between root canal pathogensand anti-infective defense mechanisms may prevent tissue destructionand initiate remodeling (122). Stem cells able to generate dentin-likestructures and odontoblast-like cells were first reported in the dentalpulp in 2000 (123). A growing number of publications have since re-ported several stem cell populations of dental origin (124–126).Because pulp-derived stem cells of teeth affected by apical periodontitiswill be increasingly compromised by inflammation, stem cell recruit-ment from the PDL of neighboring teeth may be favored (127). Underappropriate conditions, PDL-derived stem cells may be suitable forengineering bone tissue, PDL, and cementum simultaneously


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(125, 128). The remaining granulation tissue after endodontictreatment may provide the stimulus required for stem cellrecruitment, proliferation, and differentiation. Regenerative responsesare mediated by a variety of growth factors, including TGF-b andother members of the TGF-b superfamily (125, 129–131).
ConclusionInflammatory reactions in the periapical tissues in response to

endodontic infection are self-limiting in the majority of cases. Both mi-crobial influx from the infected root canal and excessive destruction oftooth-supporting structures are controlled for extended periods of timeby complex interactions between immunoinflammatory cells and solu-ble mediators. The permanent shift between anti- and proinflammatoryprocesses influences the balance between tissue destruction and regen-eration at different stages of apical periodontitis. This dynamic equilib-rium between protection from infection and host tissue injury andrepair processes is driven by overlapping networks of pleiotropicand redundant regulatory mechanisms. Recent advances have high-lighted important new aspects of overlapping ‘‘destructive’’ and ‘‘protec-tive’’ reactions in apical periodontitis. Focus on cellular and moleculartargets may allow the development and translation of novel therapeutictools tomoderate periapical tissue destruction and stimulate supportingtissue regeneration.

AcknowledgmentsThe authors thank Dr Anthony J. Smith, Birmingham, UK, and

Dr Gunnar Bergenholz, Gothenburg, Sweden, for their criticalreading of this manuscript and Erika S�ari for graphical work.

Supported by the T�AMOP 4.2.1./B-09/1/KONV-2010-0007project. The project is cofinanced by the European Union and theEuropean Social Fund and the OTKA (Hungarian Scientific ResearchFund) K 105034 grant.

The authors deny any conflicts of interest related to thisstudy.

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