Signaling Pathways Activation by Primary EndodonticInfectious Contents and Production of InflammatoryMediatorsFrederico C. Martinho, DDS, MSC, PhD,* Fabio R.M. Leite, DDS, MSc, PhD,

Wanderson M.M. Chiesa, DDS, MSc, PhD, Gustavo G. Nascimento, DDS, MPH,

Magda Feres, DDS, MSc, PhD,jj and Brenda P.F.A. Gomes, DDS, MSc, PhD

endotoxin levels. (J Endod 2014;40:484489)

Clinical Researchhttp://dx.doi.org/10.1016/j.joen.2013.10.022From the *Department of Restorative Dentistry, Endodontics Division, UNESP-UNIV Estadual Paulista, S~ao Jose dos Campos Dental School, S~ao Jose dos Campos, S~aoPaulo; Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas, UNICAMP, Piracicaba, S~ao Paulo;Department of Semiology and Clinics, Periodontics Division, Federal University of Pelotas, School of Dentistry, UFPel, Pelotas, Rio Grande do Sul; Department of Restor-ative Dentistry, Endodontics Division, Amazonas State University, Manaus, Amazonas; jjDepartment of Restorative Dentistry, Postgraduation Division, Federal Universityof Pelotas, School of Dentistry, UFPel, Pelotas, Rio Grande do Sul; and Dental Research Division, Department of Periodontology, Guarulhos University, Guarulhos, S~aoPaulo, Brazil.

Address requests for reprints to Dr Brenda P.F.A. Gomes, Endodontics Division, Department of Restorative Dentistry, Piracicaba Dental School State University ofCampinas, UNICAMP, Av Limeira 901, Bairro Areiao, Piracicaba, SP, Brazil. E-mail address: bpgomes@fop.unicamp.br0099-2399/$ - see front matter

Copyright 2014 American Association of Endodontists.levels of IL-6 and IL-10, all significantly affected by

diseases, the aims of this clinical study were to investigate the bacterial communityinvolved, to evaluate its ability to activate macrophage TLR-4 receptor through p38AbstractIntroduction: This study investigated the bacterialcommunity involved in primary endodontic diseases,evaluated its ability to activate the macrophage Toll-like receptor 4 receptor through p38 mitogen-activatedprotein kinase (MAPK) and nuclear factor kappa B(NF-kB) signaling pathways, and determined the levelsof endotoxins and interleukins (interleukin [IL]-6 and-10) produced by endodontic content-stimulated macro-phages.Methods: Samples were taken from 21 root ca-nals by using sterile/apyrogenic paper points. Raw 264.7macrophages were stimulated with root canal contents.Checkerboard DNA-DNA hybridization was used for bac-terial analysis and the limulus amebocyte lysate assayfor endotoxin measurement; p38 MAPK and NF-kB acti-vation was determined by Western blot analysis. IL-6and IL-10 were measured using the enzyme-linkedimmunosorbent assay. Results: Bacteria and endo-toxins were detected in 100% of the samples (21/21). The most frequently observed species were Parvi-monas micra (16/21, 76%), Fusobacterium nu-cleatum ssp. nucleatum (15/21, 71%), andPorphyromonas endodontalis (14/21, 66%). Cor-relations were found between endotoxins and IL-6 andIL-10 (P < .05); p38 phosphorylation had a peak at 60minutes, and NF-kB was quickly activated after 10 mi-nutes of stimulation. Conclusions: It was concludedthat the complex bacterial community was shown tobe a potent activator of TLR-4 determined by the p38MAPK and NF-kB signaling pathways, culminating in ahigh antigenicity against macrophages through the484 Martinho et al.Key WordsEndotoxin, interleukin, macrophage, root canal, signaling pathways

Bacterial infection of the dental pulp results in tissue destruction and, eventually,periapical bone resorption (1). Inflammatory cytokine production is induced inresponse to the infection, which is primarily produced by monocytes/macrophagesthat play a central role in cytokine production by modulating many aspects of the in-flammatory response (2, 3).

It has long been known that primary endodontic infection has a polymicrobial eti-ology caused by both gram-positive and gram-negative anaerobic bacteria (4, 5). Thelatter have lipopolysaccharides (LPSs, known as endotoxins) located on the outer layersof bacterial cell walls (6) and are considered one of the major factors involved in theinflammation response.

LPSs have been shown to interact with Toll-like receptors (TLRs), both TLR-2 andTLR-4, but with greater affinity for TLR-4 (7, 8), which in turn recognizes the LPSmolecule and activates multiple downstream signaling pathways (9, 10). The bindingof LPSs to TLR-4 leads to the activation of p38 mitogen-activated protein kinase (p38MAPK) (an upstream effector common to many inflammatory cytokines) and NF-kBtranscription factor (central to several immune and inflammatory responses), whichare responsible for proinflammatory cytokine production, such as interleukin (IL)-1beta, tumor necrosis factor alpha, prostaglandin E2, and IL-6 and -10 (1113).

The action of proinflammatory/bone resorptive mediators is potentially regulatedby a network of different cytokines (7, 8). This network in endodontic infection iscomplex (7) and changes according to disease destruction and activity (9). The redun-dancy and overlapping of cytokines make it difficult to understand the inflammatoryprocess (8, 10). Thus, the study of different signaling pathways involved inendodontic gene expression may allow us to gain an understanding of themodulation of the host response affecting the whole cytokine profile.

In order to better understand the immunobiology involved in primary endodonticJOE Volume 40, Number 4, April 2014

Clinical Research

MAPK and NF-kB signaling pathways, and to determine the levels ofendotoxins and interleukins (IL-6 and -10) produced by endodonticcontent-stimulated macrophages.

Materials and MethodsPatient Selection

Twenty-one patients needing endodontic treatment who attendedthe Piracicaba Dental School, Piracicaba, S~ao Paulo, Brazil, wereincluded in this study. The age of the patients ranged from 1373 years.Samples were collected from 21 root canals with pulp necrosis; they allshowed radiographic evidence of apical periodontitis. The selected teethshowed the absence of periodontal pockets more than 4 mm in depth.

A detailed dental history was obtained from each patient. Those whohad received antibiotic treatment during the past 3 months or who hadany systemic disease were excluded. The Human Research Ethics Com-mittee of the Piracicaba Dental School approved the protocol describingthe sample collection for this investigation, and all volunteer patientssigned an informed consent form before their participation in the study.

Sampling ProceduresAll materials used in this study were heat sterilized at 200C for 4

hours, thus becoming apyrogenic. The method followed for disinfectionof the operative field was described previously (3, 4). The teeth wereisolated with a rubber dam; the crown and surrounding structureswere disinfected with 30% H2O2 for 30 seconds followed by 2.5%sodium hypochlorite for a further 30 seconds. Subsequently, 5%sodium thiosulfate was used to inactivate the irrigant. The sterility ofthe external surfaces of the crown was checked by taking a swabsample from the crown surface and streaking it onto blood agarplates, which were incubated aerobically and anaerobically.

A 2-stage access cavity preparation wasmade without the use of wa-ter spray but under manual irrigation with sterile/apyrogenic saline so-lution and by using a sterile/apyrogenic high-speed diamond bur. Thefirst stage was performed to promote a major removal of contaminants.In the second stage, before entering the pulp chamber, the access cavitywas disinfected according to the protocol described previously. The ste-rility of the internal surface of the access cavity was checked as previouslydescribed, and all procedures were performed aseptically. A new sterileand apyrogenic bur was used followed by irrigation of the root canal ac-cess with sterile apyrogenic water. The endotoxin sample was taken byintroducing sterile pyrogen-free paper points (size #15; Dentsply-Maillefer, Ballaigues, Switzerland) into the full length of the canal (deter-mined radiographically) and retained in position for 60 seconds. Next,the paper point was immediately placed on a pyrogen-free glass andfrozen at80C for limulus amebocyte lysate (LAL) assay and cell cul-ture stimulation. The procedure was repeatedwith 5 sterile paper points.The paper points were pooled in a sterile tube containing 1 mL VMGA IIItransport medium (Becton Dickinson Microbiology Systems, Cockeys-ville, MD) and then immediately processed for DNA extraction to detectthe target bacteria using the checkerboard DNA-DNA technique.

Microbiological Assessment: CheckerboardDNA-DNA Hybridization

The presence, levels, and proportions of 40 bacterial species(Table 1) were determined in each sample by modifying the checker-board DNA-DNA hybridization method as described by previous investi-gations (11, 14). Microbial DNA from endodontic samples andAmerican Type Culture Collection bacterial strains (probes wereextracted and purified with QIAamp DNA Mini Kit; Qiagen, Hilden,Germany) were obtained according to the manufacturers instructions.The concentration of DNA (absorbance at 260 nm) was determinedJOE Volume 40, Number 4, April 2014with a spectrophotometer (Nanodrop 2000; Thermo Scientific,Wilmington, DE).

The samples were boiled for 10 minutes and neutralized with 0.8mL 5 mol/L ammonium acetate. The released DNA was then placed intoextended slots of a Minislot 30 apparatus (Immunetics, Cambridge,MA), concentrated onto a 15 15 positively charged nylon membrane(Boehringer, Mannheim, Germany), and fixed to the membrane by in-cubation at 120C for 20 minutes. A Miniblotter 45 (Immunetics) de-vice was used to hybridize the 40 digoxigenin-labeled whole-genomicDNA probes at right angles to the lanes of the clinical samples. Boundprobes were detected by using phosphatase-conjugated antibodies todigoxigenin and chemiluminescence (CDP-Star Detection Reagent;Amersham Biosciences, Chicago, IL). Signals were visually evaluatedby comparison with 2 standards. These standards consisted of a mixtureof 105 and 106 cells from each bacteria tested placed in the last 2 lanesof eachmembrane. The signals were coded in 6 different classes in rela-tion to the following count levels:

0: Not detected1: 106 cells

The sensitivity of this assay was adjusted to permit the detection of104 cells of a given species by adjusting the concentration of each DNAprobe.

Endotoxin Detection (LAL Assay)Determination of Endotoxin Concentration (Turbidi-metric Test and LAL Assay). The turbidimetric test (BioWhitakerInc, Walkersville, MD) was used to measure endotoxin concentrationsin root canals using the LAL technique. As a parameter for calculation ofthe amount of endotoxins in the root canal samples, a standard curvewas plotted by using the endotoxins supplied by the kit with known con-centrations (100 EU/mL) and dilutions, resulting in the following finalconcentrations according to the manufacturers instructions: 0.01,0.10, 1, and 10 EU/mL.

Test Procedure. All reactions were performed in duplicate to vali-date the test. A 96-well microplate (Corning Costar, Cambridge, MA)was used in a heat block at 37C and maintained at this temperaturethroughout the assay. First, the endotoxin samples were suspended in1 mL LAL water supplied by the kit and agitated in vortex for 60 secondsand serial diluted to 101. Next, 100 mL of the blank followed by thestandard endotoxin solutions at their concentrations (ie, 0.01, 0.10,1, and 10 EU/mL), and 100 mL of the samples were immediately addedin duplicate to the 96-well microplate. The test procedure was per-formed following the manufacturers instructions. The absorbenciesof endotoxin were measured individually by using an enzyme-linkedimmunosorbent assay (ELISA) plate reader (Ultramark; Bio-Rad Labo-ratories, Inc, Hercules, CA) at 340 nm.

Calculation of Endotoxin Concentration. Because the meanabsorbance value of the standards was directly proportional to the con-centration of endotoxins present, the endotoxin concentration wasdetermined from the standard curve.

Activation of Signaling PathwaysP38 MAPK and NF-kB Signaling Pathways (Western Blot).For these short-term experiments, 3 105 RAW 264.7 macrophageswere grown for 24 hours in each well of the 6-well plates andSignaling Pathways Activation by Endodontic Infectious Contents 485

Capnocytophaga gingivalis 33624* Propionibacterium acnes I and II 11827 and 11828*

Clinical Researchsubsequently treated with 60mL root canal contents. No stimulated cellswere used as a control group (0 minutes). Cell stimulation was main-tained for 10, 30, and 60 minutes. At the end of each experimentalperiod, whole-cell lysates were harvested by scraping the cells in so-dium dodecyl sulfate sample buffer (62.5 mmol/L Tris-HCl buffer, pH= 6.8, 10% glycerol, 50 mmol/L dithiothreitol, 2% sodium dodecyl sul-fate, 0.01% bromophenol blue) on ice followed by sonication for 10seconds and heat denaturation at 95

C for 5 minutes. The total protein

content was quantified by the Lowry method (DC Assay, Bio-Rad Labo-

Capnocytophaga ochracea 33596*Capnocytophaga sputigena 33612*Eikenella corrodens 23834*Enterococcus faecalis 29212*Eubacterium nodatum 33099*Eubacterium saburreum 33271*Fusobacterium nucleatum ssp.

polymorphum10953*

Fusobacterium nucleatum ssp.nucleatum

25586*

Fusobacterium nucleatum ssp.vicentii

49256*

Fusobacterium periodonticum 33693*Gemella morbillorum 27824*

*American Type Culture Collection.Forsyth Institute, Boston, MA.TABLE 1. Bacterial Strains Used for the Development of the DNA Probes

Species Strain

Actinomyces gerecseriae 23860*Actinomyces israelii 12102*Actinomyces naeslundii 12104*Actinomyces odontolyticus 17929*Aggregatibacter

actinomycetemcomitans43718*

Campylobacter gracilis 33236*Campylobacter rectus 33238*Campylobacter showae 51146*ratories, Inc). For Western blotting, 30 mg total protein was separatedwith 10% Tris-HCl polyacrylamide gels run at 100 V for 60 minutes andsubsequently electrotransferred to nitrocellulose membranes foranother 60 minutes in a semidry apparatus at 110 mA/gel. Membraneswere blocked (tris-buffered saline with 5% nonfat dry milk, 0.1% po-lisorbate 20) for 1 hour at room temperature and then probed withthe primary antibodies overnight at 4

C. The presence of primary anti-

bodies for phospho-p38 and NF-kB p65 (Santa Cruz Biotechnology,Santa Cruz, CA) was detected by using horseradish peroxidaseconju-gated secondary antibodies and a chemiluminescence system (Super-Signal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL).Digital images of the radiographic films exposed to the membraneswere obtained with a gel documentation system. The expression levelsof glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were deter-mined to verify equal loading of proteins.

Antigenicity of Bacterial Endodontic ContentsCell Culture/Stimulation. Macrophages (RAW 264.7) werecultured in 100-mm culture plates containing Dulbecco modified Eagleminimal essential medium supplemented with 100 IU/mL penicillin,100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serumand maintained in a humidified atmosphere at 37C and 5% CO2 until90% confluence. Unless noted otherwise, all tissue culture reagentswere obtained from Invitrogen (Carlsbad, CA). The cells were releasedfrom the 100-mm plates with 0.25% trypsin and counted in a Neubauer

486 Martinho et al.chamber, and a total of 104 macrophages were grown for 48 hoursin each well of the 6-well plates. Next, they were deinduced by incuba-tion for 8 hours in culture medium (Dulbecco modified Eagle medium)containing 0.3% fetal bovine serum and stimulated with 60 mL rootcanal contents during 24 hours in order to quantify the total amountof protein released in the culture media (ie, IL-6 and IL-10 protein).The supernatants were collected and stored at 80C until proteinevaluation.

Measurements of IL-6 and IL-10 Levels. The amounts of IL-6

Selenomonas noxia 43541*Streptococcus anginosus 33397*Streptococcus constellatus 27823*Streptococcus gordonii 10558*Streptococcus intermedius 27335*Streptococcus mitis 49456*Streptococcus oralis 35037*

Tannerella forsythia 43037*

Treponema denticola B1

Treponema socransckii S1

Veillonella parvula 10790*Species Strain

Leptotrichia bucallis 14201*Neisseria mucosa 19696*Parvimonas micra 33270*Porphyromonas endodontalis 35406*Porphyromonas gingivalis 33277*

Prevotella intermedia 25611*Prevotella melaninogenica 25845*Prevotella nigrescens 33563*and IL-10 released in the culture media after root canal content stimu-lation of RAW 264.7 macrophages were measured using the DuoSetELISA Kit (R&D, Minneapolis, MN). Medium of unstimulated macro-phage culture was used as the negative control. Standard, control, orsample solution was added to the ELISA well plate, which had been pre-coated with specific monoclonal capture antibody. After being gentlyshaken for 3 hours at room temperature, polyclonal antiIL-6 andantiIL-10 antibodies, conjugated with horseradish peroxidase, wererespectively added to the solution and then incubated for 1 hour atroom temperature. Substrate solution containing hydrogen peroxidaseand chromogen was added and allowed to react for 20 minutes. Thelevels of cytokines were assessed by a micro-ELISA reader at 450 nmand normalized with an abundance of standard solution. Each densito-metric value expressed as mean standard deviation was obtainedfrom 3 independent experiments.

Statistical AnalysisThe data collected for each case (clinical features and bacterial

detection) were typed into a spreadsheet and statistically analyzed byusing SPSS for Windows (SPSS, Inc, Chicago, IL). Bacterial prevalencewas computed by determining the proportions of root canals colonizedby each species at counts$104 cells. The Pearson chi-square test and1-sided Fisher exact test (as appropriate) were chosen to assess the nullhypothesis that there was no relationship between bacterial species. ThePearson coefficient was used to correlate the amount of LPS, IL-6, and

JOE Volume 40, Number 4, April 2014

IL-10 levels with the size of radiolucent area and levels of bacteria. Thecorrelation between the presence of clinical/radiographic findings andthe median levels of LPS, IL-6, and IL-10 was analyzed by using the Stu-dent t test or the Mann-Whitney U test. P < .05 was considered statisti-cally significant.

ResultsThe following clinical features were observed in the 21 root canals

analyzed: pain on palpation (9/21), tenderness to percussion (8/21),presence of exudation (12/21), and radiolucent area $2 mm (11/21) and 104 was 12.23 2.44 (ranging from 816 bacterial species). The most frequentlydetected species were Parvimonas micra (16/21, 76%), Fusobacte-rium nucleatum ssp. nucleatum (15/21, 71%), Porphyromonasendodontalis (14/21, 66%), F. nucleatum spp. vicentii (13/21,61%), Prevotella nigrescens (12/21, 57%), Leptotrichia bucallis(12/21, 57%), and F. nucleatum ssp. polymorphum (12/21,57%). The frequency (absolute number of root canals colonized[indicated by bars]) and DNA concentration (106 [indicated by legend]) of individual bacterial speciesinvestigated are shown in Figure 1.

Determination of Endotoxin Concentration(Turbidimetric Test and LAL Assay)

Endotoxin was detected in 100% of the root canals investigated(21/21) with a median value of 7.490 pg/mL ranging from 27289.000 pg/mL.

Activation of p38 and NF-kB Signaling Pathwaysby Endodontic Contents in Macrophage Cultures

RAW 264.7 cultures were stimulated with endodontic root canalcontent for 0 (unstimulated control), 10, 30, and 60 minutes. Thep38 pathway tended to show a slight increase in activation after 60 mi-nutes of cell stimulation. NF-kBwas quickly activated after 10minutes ofstimulation with a peak at 30 minutes, and the activation was sustainedeven at 60 minutes of analysis (Fig. 2).

Antigenicity of Bacterial Endodontic Contents (CellCulture/Stimulation and Cytokine Expression)

IL-6 and IL-10 were detected in all culture media after stimulationwith root canal contents. In unstimulated cultures, no detectable levels ofboth cytokines were found. The median levels recorded for IL-6 and IL-10 were 270.151 pg/mL (range, 146.674365.017 pg/mL) and 39.997pg/mL (range, 17.28150.111 pg/mL), respectively (Table 2). IL-6levels from cell cultures stimulated with material from teeth with tender-ness to percussion (283.488 pg/mL) were significantly higher than in itsabsence (225.729) (P < .05) (Table 2). Thus, a higher level of IL-10was found in macrophage stimulated with contents from teeth withpain on palpation (41.987 pg/mL) than in those without it (35.520pg/mL) (P < .05). Relatively higher levels of IL-6 and IL-10 were foundwhen macrophages were stimulated with contents from teeth showing aradiolucent area$2 mm (IL-6 = 279.401 pg/mL and IL-10 = 40.992

by

Clinical ResearchFigure 1. The frequency (absolute number of root canals colonized [indicatedand >106 [indicated by legend) of individual bacterial species investigated.JOE Volume 40, Number 4, April 2014bars]) and DNA concentration (1 =

pg/mL) compared with those

With regard to the downstream of TLR-4 activation, the phosphor-ylation of IKB kinase (IKK) complex requires the recruitment of tumornecrosis factor receptorassociated factor (TRAF) 2 and TRAF5, whichwill recruit receptor-interacting protein to act as a scaffold for the bind-ing of the IKK complex (30, 31). TLR signaling requires a different set ofadaptors, including myeloid differentiation factor 88 (MyD88) and

8. Stashenko P, Teles R, DSouza R. Periapical inflammatory responses and their mod-ulation. Crit Rev Oral Biol Med 1998;9:498521.

9. Garcia de Aquino S, Manzolli Leite FR, Stach-Machado DR, et al. Signaling path-ways associated with the expression of inflammatory mediators activated duringthe course of two models of experimental periodontitis. Life Sci 2009;84:74554.

10. Yamazaki K, Nakajima T, Gemmell E, et al. IL-4- and IL-6-producing cells in humanperiodontal disease tissue. J Oral Pathol Med 1994;23:34753.

Clinical ResearchTIR domain-containing adaptor protein (TIRAP), which will recruitinterleukin 1 receptorassociated kinase 1 (IRAK1) or TRAF6, and itis currently believed that the kinase transforming growth factor betaactivated kinase 1 (TAK1) would link TRAF6 to the IKK complex, thusculminating in NF-kB translocation to the nucleus (30, 31). The p38pathway requires different adaptors in which TAK1 recruitstransforming growth factor betaactivated kinase 1/MAP3K7-bindingprotein (TAB)-1 and TAB-2. TAB 1/2 induces mitogen-activated proteinkinase 3 and 6 phosphorylation (MAP3K6), which phosphorylates p38at Thr180 and Tyr182 (32). These characteristics of the signalingpathway downstream of different receptorsmay explain the temporal dif-ferences in NF-kB and p38 activation.

Overall, the primary endodontic infectious contents comprised by acomplex bacterial community was shown to be a potent activator of TLR-4, determined by p38MAPK andNF-kB signaling pathways, culminating ina high antigenicity against macrophages through the levels of IL-6 and IL-10; all were significantly affected by endotoxin levels and participated inthe clinical findings involved in endodontic disease. Further studiesshould be performed in order to evaluate the effect of root canal therapieson infection control of bacterial and fungi endodontic contents throughthe measurement of cytokines production. Also, p38 and NF-kB or thecomponents of their pathways can be a therapeutic target for the devel-opment of new drugs against inflammatory root canal diseases.

AcknowledgmentsThe authors would like to thank Ana Regina de Oliveira Polay

for her technical assistance. They also are thankful to Lonza for theKinetic-QCL equipment.

Supported by the Brazilian agencies FAPESP (10/19136-1; 10/17877-4; 13/02402-9), CNPq (302575/2009-0; 150557/2011-6),and CAPES.

The authors deny any conflicts of interest.

References1. Bergenholtz G. Pathogenic mechanisms in pulpal disease. J Endod 1990;16:

98101.2. Beutler B, Cerami A. The biology of cachectin/TNFa primary mediator of the host

response. Annu Rev Immunol 1989;7:62555.3. Martinho FC, Chiesa WM, Leite FR, et al. Antigenic activity of bacterial endodontic

contents from primary root canal infection with periapical lesions against macro-phage in the release of interleukin-1beta and tumor necrosis factor alpha.J Endod 2010;36:146774.

4. Gomes BP, Pinheiro ET, Gade-Neto CR, et al. Microbiological examination of in-fected dental root canals. Oral Microbiol Immunol 2004;19:716.

5. Rocas IN, Siqueira JF Jr. Identification of bacteria enduring endodontic treatmentprocedures by a combined reverse transcriptase-polymerase chain reaction andreverse-capture checkerboard approach. J Endod 2010;36:4552.

6. Schein B, Schilder H. Endotoxin content in endodontically involved teeth. J Endod1975;1:1921.

7. Martinho FC, Chiesa WM, Leite FR, et al. Correlation between clinical/radiographicfeatures and inflammatory cytokine networks produced by macrophages stimulatedwith endodontic content. J Endod 2012;38:7405.JOE Volume 40, Number 4, April 201411. Socransky SS, Smith C, Martin L, et al. Checkerboard DNA-DNA hybridization. Bio-techniques 1994;17:78892.

12. Oliveira LD, Carvalho CA, Carvalho AS, et al. Efficacy of endodontic treatment forendotoxin reduction in primarily infected root canals and evaluation of cytotoxiceffects. J Endod 2012;38:10537.

13. Jacinto RC, Gomes BP, Ferraz CC, et al. Microbiological analysis of infected root ca-nals from symptomatic and asymptomatic teeth with periapical periodontitis and theantimicrobial susceptibility of some isolated anaerobic bacteria. Oral Microbiol Im-munol 2003;18:28592.

14. Vianna ME, Horz HP, Conrads G, et al. Comparative analysis of endodontic patho-gens using checkerboard hybridization in relation to culture. Oral Microbiol Immu-nol 2008;23:28290.

15. Poltorak A, Smirnova I, He X, et al. Genetic and physical mapping of the Lps locus:identification of the toll-4 receptor as a candidate gene in the critical region. BloodCells Mol Dis 1998;24:34055.

16. Hoshino K, Takeuchi O, Kawai T, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 asthe Lps gene product. J Immunol 1999;162:374952.

17. Riva M, Kallberg E, Bjork P, et al. Induction of nuclear factor-kappaB responses bythe S100A9 protein is Toll-like receptor-4-dependent. Immunology 2012;137:17282.

18. Zheng W, Zheng X, Liu S, et al. TNFalpha and IL-1beta are mediated by both TLR4and Nod1 pathways in the cultured HAPI cells stimulated by LPS. Biochem BiophysRes Commun 2012;420:7627.

19. Sassone LM, Fidel RA, Faveri M, et al. A microbiological profile of unexposed andexposed pulp space of primary endodontic infections by checkerboard DNA-DNAhybridization. J Endod 2012;38:88993.

20. Moraes SR, Siqueira JF Jr, Colombo AP, et al. Comparison of the effectiveness of bac-terial culture, 16S rDNA directed polymerase chain reaction, and checkerboardDNA-dNA hybridization for detection of Fusobacterium nucleatum in endodontic in-fections. J Endod 2002;28:869.

21. Martinho FC, Chiesa WM, Leite FR, et al. Antigenicity of primary endodontic infec-tion against macrophages by the levels of PGE(2) production. J Endod 2011;37:6027.

22. Ferwerda G, Meyer-Wentrup F, Kullberg BJ, et al. Dectin-1 synergizes with TLR2 andTLR4 for cytokine production in human primary monocytes and macrophages. CellMicrobiol 2008;10:205866.

23. Gomes BP, Endo MS, Martinho FC. Comparison of endotoxin levels found in primaryand secondary endodontic infections. J Endod 2012;38:10826.

24. Fong Y, Moldawer LL, Marano M, et al. Endotoxemia elicits increased circulatingbeta 2-IFN/IL-6 in man. J Immunol 1989;142:23214.

25. Huang GT, Do M, Wingard M, et al. Effect of interleukin-6 deficiency on the forma-tion of periapical lesions after pulp exposure in mice. Oral Surg Oral Med OralPathol Oral Radiol Endod 2001;92:838.

26. Tinsley JH, South S, Chiasson VL, et al. Interleukin-10 reduces inflammation, endo-thelial dysfunction, and blood pressure in hypertensive pregnant rats. Am J PhysiolRegul Integr Comp Physiol 2010;298:R7139.

27. Ono K, Han J. The p38 signal transduction pathway: activation and function. CellSignal 2000;12:113.

28. ONeill LA. The interleukin-1 receptor/Toll-like receptor superfamily: 10 years ofprogress. Immunol Rev 2008;226:108.

29. Albiger B, Dahlberg S, Henriques-Normark B, et al. Role of the innate immune sys-tem in host defence against bacterial infections: focus on the Toll-like receptors.J Intern Med 2007;261:51128.

30. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004;18:2195224.31. Tergaonkar V. NFkappaB pathway: a good signaling paradigm and therapeutic

target. Int J Biochem Cell Biol 2006;38:164753.32. Kumar S, Jiang MS, Adams JL, et al. Pyridinylimidazole compound SB 203580 in-

hibits the activity but not the activation of p38 mitogen-activated protein kinase. Bio-chem Biophys Res Commun 1999;263:82531.Signaling Pathways Activation by Endodontic Infectious Contents 489

Signaling Pathways Activation by Primary Endodontic Infectious Contents and Production of Inflammatory MediatorsMaterials and MethodsPatient SelectionSampling ProceduresMicrobiological Assessment: Checkerboard DNA-DNA HybridizationEndotoxin Detection (LAL Assay)Determination of Endotoxin Concentration (Turbidimetric Test and LAL Assay)Test ProcedureCalculation of Endotoxin Concentration

Activation of Signaling PathwaysP38 MAPK and NF-kB Signaling Pathways (Western Blot)

Antigenicity of Bacterial Endodontic ContentsCell Culture/StimulationMeasurements of IL-6 and IL-10 Levels

Statistical Analysis

ResultsMicrobiological Assessment: Checkerboard DNA-DNA HybridizationDetermination of Endotoxin Concentration (Turbidimetric Test and LAL Assay)Activation of p38 and NF-B Signaling Pathways by Endodontic Contents in Macrophage CulturesAntigenicity of Bacterial Endodontic Contents (Cell Culture/Stimulation and Cytokine Expression)

DiscussionAcknowledgmentsReferences