Clinical Research

Signaling Pathways Activation by Primary Endodontic Infectious Contents and Production of Inammatory Mediators
Frederico 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
Abstract
Introduction: This study investigated the bacterial community involved in primary endodontic diseases, evaluated its ability to activate the macrophage Tolllike receptor 4 receptor through p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) signaling pathways, and determined the levels of endotoxins and interleukins (interleukin [IL]-6 and -10) produced by endodontic content-stimulated macrophages. Methods: Samples were taken from 21 root canals by using sterile/apyrogenic paper points. Raw 264.7 macrophages were stimulated with root canal contents. Checkerboard DNA-DNA hybridization was used for bacterial analysis and the limulus amebocyte lysate assay for endotoxin measurement; p38 MAPK and NF-kB activation was determined by Western blot analysis. IL-6 and IL-10 were measured using the enzyme-linked immunosorbent assay. Results: Bacteria and endotoxins were detected in 100% of the samples (21/ 21). The most frequently observed species were Parvimonas micra (16/21, 76%), Fusobacterium nucleatum ssp. nucleatum (15/21, 71%), and Porphyromonas endodontalis (14/21, 66%). Correlations were found between endotoxins and IL-6 and IL-10 (P < .05); p38 phosphorylation had a peak at 60 minutes, and NF-kB was quickly activated after 10 minutes of stimulation. Conclusions: It was concluded that the complex bacterial community was shown to be a potent activator of TLR-4 determined by the p38 MAPK and NF-kB signaling pathways, culminating in a high antigenicity against macrophages through the levels of IL-6 and IL-10, all signicantly affected by endotoxin levels. (J Endod 2014;40:484489)

Key Words
Endotoxin, interleukin, macrophage, root canal, signaling pathways

acterial infection of the dental pulp results in tissue destruction and, eventually, periapical bone resorption (1). Inammatory cytokine production is induced in response to the infection, which is primarily produced by monocytes/macrophages that play a central role in cytokine production by modulating many aspects of the inammatory response (2, 3). It has long been known that primary endodontic infection has a polymicrobial etiology caused by both gram-positive and gram-negative anaerobic bacteria (4, 5). The latter have lipopolysaccharides (LPSs, known as endotoxins) located on the outer layers of bacterial cell walls (6) and are considered one of the major factors involved in the inammation response. LPSs have been shown to interact with Toll-like receptors (TLRs), both TLR-2 and TLR-4, but with greater afnity for TLR-4 (7, 8), which in turn recognizes the LPS molecule and activates multiple downstream signaling pathways (9, 10). The binding of LPSs to TLR-4 leads to the activation of p38 mitogen-activated protein kinase (p38 MAPK) (an upstream effector common to many inammatory cytokines) and NF-kB transcription factor (central to several immune and inammatory responses), which are responsible for proinammatory cytokine production, such as interleukin (IL)-1 beta, tumor necrosis factor alpha, prostaglandin E2, and IL-6 and -10 (1113). The action of proinammatory/bone resorptive mediators is potentially regulated by a network of different cytokines (7, 8). This network in endodontic infection is complex (7) and changes according to disease destruction and activity (9). The redundancy and overlapping of cytokines make it difcult to understand the inammatory process (8, 10). Thus, the study of different signaling pathways involved in endodontic gene expression may allow us to gain an understanding of the modulation of the host response affecting the whole cytokine prole. In order to better understand the immunobiology involved in primary endodontic diseases, the aims of this clinical study were to investigate the bacterial community involved, to evaluate its ability to activate macrophage TLR-4 receptor through p38

From the *Department of Restorative Dentistry, Endodontics Division, UNESP-UNIV Estadual Paulista, S~ ao Jos e dos Campos Dental School, S~ ao Jos e dos Campos, S~ ao ao Paulo; Paulo; Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas, UNICAMP, Piracicaba, S~ Department of Semiology and Clinics, Periodontics Division, Federal University of Pelotas, School of Dentistry, UFPel, Pelotas, Rio Grande do Sul; Department of Restorative Dentistry, Endodontics Division, Amazonas State University, Manaus, Amazonas; jjDepartment of Restorative Dentistry, Postgraduation Division, Federal University ao of Pelotas, School of Dentistry, UFPel, Pelotas, Rio Grande do Sul; and Dental Research Division, Department of Periodontology, Guarulhos University, Guarulhos, S~ Paulo, Brazil. Address requests for reprints to Dr Brenda P.F.A. Gomes, Endodontics Division, Department of Restorative Dentistry, Piracicaba Dental School State University of Campinas, UNICAMP, Av Limeira 901, Bairro Areiao, Piracicaba, SP, Brazil. E-mail address: bpgomes@fop.unicamp.br 0099-2399/$ - see front matter Copyright 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.10.022

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MAPK and NF-kB signaling pathways, and to determine the levels of endotoxins and interleukins (IL-6 and -10) produced by endodontic content-stimulated macrophages. with a spectrophotometer (Nanodrop 2000; Thermo Scientic, Wilmington, DE). The samples were boiled for 10 minutes and neutralized with 0.8 mL 5 mol/L ammonium acetate. The released DNA was then placed into extended slots of a Minislot 30 apparatus (Immunetics, Cambridge, MA), concentrated onto a 15 15 positively charged nylon membrane (Boehringer, Mannheim, Germany), and xed to the membrane by incubation at 120 C for 20 minutes. A Miniblotter 45 (Immunetics) device was used to hybridize the 40 digoxigenin-labeled whole-genomic DNA probes at right angles to the lanes of the clinical samples. Bound probes were detected by using phosphatase-conjugated antibodies to digoxigenin and chemiluminescence (CDP-Star Detection Reagent; Amersham Biosciences, Chicago, IL). Signals were visually evaluated by comparison with 2 standards. These standards consisted of a mixture of 105 and 106 cells from each bacteria tested placed in the last 2 lanes of each membrane. The signals were coded in 6 different classes in relation to the following count levels: 0: 1: 2: 3: 4: 5: Not detected <105 cells Nearly 105 cells Between 105 and 106 cells Approximately 106 cells >106 cells

Materials and Methods

Patient Selection Twenty-one patients needing endodontic treatment who attended the Piracicaba Dental School, Piracicaba, S~ ao Paulo, Brazil, were included in this study. The age of the patients ranged from 1373 years. Samples were collected from 21 root canals with pulp necrosis; they all showed radiographic evidence of apical periodontitis. The selected teeth showed the absence of periodontal pockets more than 4 mm in depth. A detailed dental history was obtained from each patient. Those who had received antibiotic treatment during the past 3 months or who had any systemic disease were excluded. The Human Research Ethics Committee of the Piracicaba Dental School approved the protocol describing the sample collection for this investigation, and all volunteer patients signed an informed consent form before their participation in the study. Sampling Procedures All materials used in this study were heat sterilized at 200 C for 4 hours, thus becoming apyrogenic. The method followed for disinfection of the operative eld was described previously (3, 4). The teeth were isolated with a rubber dam; the crown and surrounding structures were 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 of the external surfaces of the crown was checked by taking a swab sample from the crown surface and streaking it onto blood agar plates, which were incubated aerobically and anaerobically. A 2-stage access cavity preparation was made without the use of water spray but under manual irrigation with sterile/apyrogenic saline solution and by using a sterile/apyrogenic high-speed diamond bur. The rst stage was performed to promote a major removal of contaminants. In the second stage, before entering the pulp chamber, the access cavity was disinfected according to the protocol described previously. The sterility of the internal surface of the access cavity was checked as previously described, and all procedures were performed aseptically. A new sterile and apyrogenic bur was used followed by irrigation of the root canal access with sterile apyrogenic water. The endotoxin sample was taken by introducing sterile pyrogen-free paper points (size #15; DentsplyMaillefer, Ballaigues, Switzerland) into the full length of the canal (determined radiographically) and retained in position for 60 seconds. Next, the paper point was immediately placed on a pyrogen-free glass and frozen at 80 C for limulus amebocyte lysate (LAL) assay and cell culture stimulation. The procedure was repeated with 5 sterile paper points. The paper points were pooled in a sterile tube containing 1 mL VMGA III transport medium (Becton Dickinson Microbiology Systems, Cockeysville, MD) and then immediately processed for DNA extraction to detect the target bacteria using the checkerboard DNA-DNA technique. Microbiological Assessment: Checkerboard DNA-DNA Hybridization The presence, levels, and proportions of 40 bacterial species (Table 1) were determined in each sample by modifying the checkerboard DNA-DNA hybridization method as described by previous investigations (11, 14). Microbial DNA from endodontic samples and American Type Culture Collection bacterial strains (probes were extracted and puried with QIAamp DNA Mini Kit; Qiagen, Hilden, Germany) were obtained according to the manufacturers instructions. The concentration of DNA (absorbance at 260 nm) was determined
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The sensitivity of this assay was adjusted to permit the detection of 104 cells of a given species by adjusting the concentration of each DNA probe.

Endotoxin Detection (LAL Assay) Determination of Endotoxin Concentration (Turbidimetric Test and LAL Assay). The turbidimetric test (BioWhitaker Inc, Walkersville, MD) was used to measure endotoxin concentrations in root canals using the LAL technique. As a parameter for calculation of the amount of endotoxins in the root canal samples, a standard curve was plotted by using the endotoxins supplied by the kit with known concentrations (100 EU/mL) and dilutions, resulting in the following nal concentrations according to the manufacturers instructions: 0.01, 0.10, 1, and 10 EU/mL. Test Procedure. All reactions were performed in duplicate to validate the test. A 96-well microplate (Corning Costar, Cambridge, MA) was used in a heat block at 37 C and maintained at this temperature throughout the assay. First, the endotoxin samples were suspended in 1 mL LAL water supplied by the kit and agitated in vortex for 60 seconds and serial diluted to 101. Next, 100 mL of the blank followed by the standard endotoxin solutions at their concentrations (ie, 0.01, 0.10, 1, and 10 EU/mL), and 100 mL of the samples were immediately added in duplicate to the 96-well microplate. The test procedure was performed following the manufacturers instructions. The absorbencies of endotoxin were measured individually by using an enzyme-linked immunosorbent assay (ELISA) plate reader (Ultramark; Bio-Rad Laboratories, Inc, Hercules, CA) at 340 nm. Calculation of Endotoxin Concentration. Because the mean absorbance value of the standards was directly proportional to the concentration of endotoxins present, the endotoxin concentration was determined from the standard curve. Activation of Signaling Pathways P38 MAPK and NF-kB Signaling Pathways (Western Blot). For these short-term experiments, 3 105 RAW 264.7 macrophages were grown for 24 hours in each well of the 6-well plates and
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TABLE 1. Bacterial Strains Used for the Development of the DNA Probes Species
Actinomyces gerecseriae Actinomyces israelii Actinomyces naeslundii Actinomyces odontolyticus Aggregatibacter actinomycetemcomitans Campylobacter gracilis Campylobacter rectus Campylobacter showae Capnocytophaga gingivalis Capnocytophaga ochracea Capnocytophaga sputigena Eikenella corrodens Enterococcus faecalis Eubacterium nodatum Eubacterium saburreum Fusobacterium nucleatum ssp. polymorphum Fusobacterium nucleatum ssp. nucleatum Fusobacterium nucleatum ssp. vicentii Fusobacterium periodonticum Gemella morbillorum
*American Type Culture Collection. Forsyth Institute, Boston, MA.

Strain
23860* 12102* 12104* 17929* 43718* 33236* 33238* 51146* 33624* 33596* 33612* 23834* 29212* 33099* 33271* 10953* 25586* 49256* 33693* 27824*

Species
Leptotrichia bucallis Neisseria mucosa Parvimonas micra Porphyromonas endodontalis Porphyromonas gingivalis Prevotella intermedia Prevotella melaninogenica Prevotella nigrescens Propionibacterium acnes I and II Selenomonas noxia Streptococcus anginosus Streptococcus constellatus Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus oralis Tannerella forsythia Treponema denticola Treponema socransckii Veillonella parvula

Strain
14201* 19696* 33270* 35406* 33277* 25611* 25845* 33563* 11827 and 11828* 43541* 33397* 27823* 10558* 27335* 49456* 35037* 43037* B1 S1 10790*

subsequently treated with 60 mL root canal contents. No stimulated cells were used as a control group (0 minutes). Cell stimulation was maintained for 10, 30, and 60 minutes. At the end of each experimental period, whole-cell lysates were harvested by scraping the cells in sodium dodecyl sulfate sample buffer (62.5 mmol/L Tris-HCl buffer, pH = 6.8, 10% glycerol, 50 mmol/L dithiothreitol, 2% sodium dodecyl sulfate, 0.01% bromophenol blue) on ice followed by sonication for 10 seconds and heat denaturation at 95 C for 5 minutes. The total protein content was quantied by the Lowry method (DC Assay, Bio-Rad Laboratories, Inc). For Western blotting, 30 mg total protein was separated with 10% Tris-HCl polyacrylamide gels run at 100 V for 60 minutes and subsequently electrotransferred to nitrocellulose membranes for another 60 minutes in a semidry apparatus at 110 mA/gel. Membranes were blocked (tris-buffered saline with 5% nonfat dry milk, 0.1% polisorbate 20) for 1 hour at room temperature and then probed with  the primary antibodies overnight at 4 C. The presence of primary antibodies for phospho-p38 and NF-kB p65 (Santa Cruz Biotechnology, Santa Cruz, CA) was detected by using horseradish peroxidaseconjugated secondary antibodies and a chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL). Digital images of the radiographic lms exposed to the membranes were obtained with a gel documentation system. The expression levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined to verify equal loading of proteins.

chamber, and a total of 104 macrophages were grown for 48 hours in each well of the 6-well plates. Next, they were deinduced by incubation for 8 hours in culture medium (Dulbecco modied Eagle medium) containing 0.3% fetal bovine serum and stimulated with 60 mL root canal contents during 24 hours in order to quantify the total amount of protein released in the culture media (ie, IL-6 and IL-10 protein). The supernatants were collected and stored at 80 C until protein evaluation. Measurements of IL-6 and IL-10 Levels. The amounts of IL-6 and IL-10 released in the culture media after root canal content stimulation of RAW 264.7 macrophages were measured using the DuoSet ELISA Kit (R&D, Minneapolis, MN). Medium of unstimulated macrophage culture was used as the negative control. Standard, control, or sample solution was added to the ELISA well plate, which had been precoated with specic monoclonal capture antibody. After being gently shaken for 3 hours at room temperature, polyclonal antiIL-6 and antiIL-10 antibodies, conjugated with horseradish peroxidase, were respectively added to the solution and then incubated for 1 hour at room temperature. Substrate solution containing hydrogen peroxidase and chromogen was added and allowed to react for 20 minutes. The levels of cytokines were assessed by a micro-ELISA reader at 450 nm and normalized with an abundance of standard solution. Each densitometric value expressed as mean standard deviation was obtained from 3 independent experiments.

Antigenicity of Bacterial Endodontic Contents Cell Culture/Stimulation. Macrophages (RAW 264.7) were cultured in 100-mm culture plates containing Dulbecco modied Eagle minimal essential medium supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum and maintained in a humidied atmosphere at 37 C and 5% CO2 until 90% conuence. Unless noted otherwise, all tissue culture reagents were obtained from Invitrogen (Carlsbad, CA). The cells were released from the 100-mm plates with 0.25% trypsin and counted in a Neubauer
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Statistical Analysis The data collected for each case (clinical features and bacterial detection) were typed into a spreadsheet and statistically analyzed by using SPSS for Windows (SPSS, Inc, Chicago, IL). Bacterial prevalence was computed by determining the proportions of root canals colonized by each species at counts $104 cells. The Pearson chi-square test and 1-sided Fisher exact test (as appropriate) were chosen to assess the null hypothesis that there was no relationship between bacterial species. The Pearson coefcient was used to correlate the amount of LPS, IL-6, and
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IL-10 levels with the size of radiolucent area and levels of bacteria. The correlation between the presence of clinical/radiographic ndings and the median levels of LPS, IL-6, and IL-10 was analyzed by using the Student t test or the Mann-Whitney U test. P < .05 was considered statistically signicant.

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 27 289.000 pg/mL. Activation of p38 and NF-kB Signaling Pathways by Endodontic Contents in Macrophage Cultures RAW 264.7 cultures were stimulated with endodontic root canal content for 0 (unstimulated control), 10, 30, and 60 minutes. The p38 pathway tended to show a slight increase in activation after 60 minutes of cell stimulation. NF-kB was quickly activated after 10 minutes of stimulation with a peak at 30 minutes, and the activation was sustained even at 60 minutes of analysis (Fig. 2). Antigenicity of Bacterial Endodontic Contents (Cell Culture/Stimulation and Cytokine Expression) IL-6 and IL-10 were detected in all culture media after stimulation with root canal contents. In unstimulated cultures, no detectable levels of both cytokines were found. The median levels recorded for IL-6 and IL10 were 270.151 pg/mL (range, 146.674365.017 pg/mL) and 39.997 pg/mL (range, 17.28150.111 pg/mL), respectively (Table 2). IL-6 levels from cell cultures stimulated with material from teeth with tenderness to percussion (283.488 pg/mL) were signicantly higher than in its absence (225.729) (P < .05) (Table 2). Thus, a higher level of IL-10 was found in macrophage stimulated with contents from teeth with pain on palpation (41.987 pg/mL) than in those without it (35.520 pg/mL) (P < .05). Relatively higher levels of IL-6 and IL-10 were found when macrophages were stimulated with contents from teeth showing a radiolucent area $2 mm (IL-6 = 279.401 pg/mL and IL-10 = 40.992

Results
The 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 <2 mm (10/21).

Microbiological Assessment: Checkerboard DNA-DNA Hybridization All root canals investigated showed bacterial signals for at least 1 of the 40 DNA bacterial probes tested. The results of the checkerboard DNA-DNA analysis revealed that 38 of the 40 DNA bacterial probes tested were reactive with at least 1 or more clinical samples. The mean number of species (standard errors of the means) detected in the 21 root canal samples at a threshold of >104 was 12.23 2.44 (ranging from 816 bacterial species). The most frequently detected species were Parvimonas micra (16/21, 76%), Fusobacterium nucleatum ssp. nucleatum (15/21, 71%), Porphyromonas endodontalis (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 (<105, 105, 105106, 106, and >106 [indicated by legend]) of individual bacterial species investigated are shown in Figure 1.

Figure 1. The frequency (absolute number of root canals colonized [indicated by bars]) and DNA concentration (1 = <105, 2 = 105, 3 = 105, 4 = 106, 5 = 106 and >106 [indicated by legend) of individual bacterial species investigated.

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The present study has revealed a diverse bacterial community involved in primarily infected root canals with a predominance of P. micra, F. nucleatum ssp. nucleatum, and P. endodontalis, thus corroborating previous investigations (2, 21). The way this combination of different bacterial species found in primarily infected root canals activates the host response is still unknown. However, it is known that bacteria can compete by the same receptor or activate different receptors by exacerbating or reducing the transcription of proinammatory proteins (22). Gram-negative bacterial endotoxins were present in 100% of the root canal contents from primarily infected teeth, also corroborating previous investigations (2, 21, 23). The ELISA assay showed that these toxins found in primary root canal infection are potent stimuli for IL-6 and IL-10 productions by macrophage cells. IL-6 and IL-10 were detected in all culture media after stimulation with root canal contents obtained from teeth with apical periodontitis, with median values of 270.151 pg/mL and 39.997 pg/mL, respectively. The median level of IL-6 in teeth with tenderness to percussion was signicantly higher than in its absence. Thus, a higher level of IL-10 was found in teeth with pain on palpation than in those without it. Such ndings indicate that IL-6 and IL-10 are intimately related to the presence of tenderness to percussion and pain on palpation, both indicative of inammation in periodontal ligament. Higher levels of endotoxin found in root canal infections were followed by an increased production of IL-6 and IL-10 by macrophages cells, indicating that the production of these cytokines may be directly promoted by endotoxin molecules (24). Therefore, not only endotoxins were related to the size of radiolucent area, which is in agreement with previous studies (2, 23), but also relatively higher levels of IL-6 and IL10 were found in teeth with a larger area of bone destruction. It should also be pointed out that the networks between different cytokines seem to regulate the formation of apical periodontitis (7, 8, 25). Although IL-6 acts as a proinammatory cytokine during periodontitis and also stimulates osteoclastic differentiation and bone resorption in chronic inammatory periodontitis (8, 25), IL-10 is actively engaged in the down-regulation of the inammatory response, thus minimizing the damage in response to microbial challenge as well as the lethal effects of LPSs (26). Thereby, IL-10 inhibits NF-kB, which consequently results in the reduction of proinammatory cytokines IL-1 beta, IL-6, and tumor necrosis factor alpha (26). The activation of TLR by infectious contents from primarily infected root canals resulted in the activation of p38 and NF-kB pathways, which are responsible for proinammatory cytokines and metalloproteinase production. These, in turn, control connective tissue remodeling in the pathological condition and many other inammatory-related molecules (9). Also, the p38 pathway participates in the regulation of immune cell proliferation and differentiation through granulocytemacrophage colony-stimulating factor (GM-CSF), colony-stimulating factor (CSF), erythropoietin (EPO), and CD40 expression (27). In the present study, intracellular pathways were distinctly activated. The p38 pathway showed a peak at 60 minutes of cell stimulation. NF-kB was quickly activated after 10 minutes of stimulation with a peak at 30 minutes, with its activation being sustained even at 60 minutes of analysis. As expected, NF-kB was quickly recruited after TLR-4 activation because it is responsible for IL-1 production, which will activate lymphocytes proliferation and cause fever (28). Also, it can be activated by both canonic and noncanonic pathways that modulate intensity and maintenance of the activation. Differences in p38 and NF-kB kinetics of activation are caused by the number of upstream adaptors and ramications in the pathways (9). NF-kB is an abundantly expressed transcription factor that is central to several immune and inammatory responses, leading to the rapid induction of cytokine secretion (29).
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Figure 2. The activation of pp38 and NF-kB p65 signaling pathways by endodontic contents in macrophage cultures; RAW 264.7 at 0, 10, 30, and 60 minutes.

pg/mL) compared with those <2 mm (IL-6 = 265.418 pg/mL and IL-10 = 37.427 pg/mL) (Table 2). Correlations were found between endotoxin contents and levels of IL-6 (P < .05, Pearson r = 0.016) and IL-10 (P < .05, Pearson r = 0.045) released in the culture media after cell stimulation.

Discussion
Data obtained in the present study revealed that bacterial endodontic contents from primarily infected root canals are potent activators of TLR determined by activation of the p38 MAPK and NF-kB signaling pathways. Additionally, TLR-4 activation culminated in the production of IL-6 and IL-10 proinammatory cytokines; both were related to the presence of specic clinical features involved in primary endodontic disease. Differently from previous investigations that focused on TLR activation and its downstream signaling pathways stimulated by puried and isolated microbial components such as LPSs (1518), the present study stimulated macrophage cells with material collected from primarily infected root canals whose contents are currently thought to be a cocktail of gram-positive and gram-negative bacterial species (4, 5). In the present study, the checkerboard DNA-DNA hybridization method using whole-genomic DNA probes allowed for simultaneous identication of a multitude of bacterial species in root canal samples, even those at low levels. Additionally, this assay has the clear advantage of allowing the bacterial species (19, 20) to be quantied because the degree of severity of an endodontic infection is related not only to the presence of pathogens but also to the number of these organisms in the infected site.
TABLE 2. The Concentration of IL-6/IL-10 in pg/mL According to the Clinical and Radiographic Findings Found in 21 Root Canals with Primary Endodontic Infection and Apical Periodontitis IL-6 (pg/mL) Median values
All teeth (21) Size of radiolucent area $2 mm (11/21) <2 mm (10/21) Tenderness to percussion Presence (8/21) Absence (13/21) Pain on palpation Presence (9/21) Absence (12/21) Exudation Presence (12/21) Absence (9/21)
*P < .05.

IL-10 (pg/mL) Median values

39.997 40.992 37.427 40.992 39.334 41.987* 35.520* 41.498 39.334

270.151 279.401 265.418 283.488* 225.729* 289.267 281.445 285.854 263.590

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With regard to the downstream of TLR-4 activation, the phosphorylation of IKB kinase (IKK) complex requires the recruitment of tumor necrosis factor receptorassociated factor (TRAF) 2 and TRAF5, which will recruit receptor-interacting protein to act as a scaffold for the binding of the IKK complex (30, 31). TLR signaling requires a different set of adaptors, including myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor protein (TIRAP), which will recruit interleukin 1 receptorassociated kinase 1 (IRAK1) or TRAF6, and it is currently believed that the kinase transforming growth factor beta activated kinase 1 (TAK1) would link TRAF6 to the IKK complex, thus culminating in NF-kB translocation to the nucleus (30, 31). The p38 pathway requires different adaptors in which TAK1 recruits transforming growth factor betaactivated kinase 1/MAP3K7-binding protein (TAB)-1 and TAB-2. TAB 1/2 induces mitogen-activated protein kinase 3 and 6 phosphorylation (MAP3K6), which phosphorylates p38 at Thr180 and Tyr182 (32). These characteristics of the signaling pathway downstream of different receptors may explain the temporal differences in NF-kB and p38 activation. Overall, the primary endodontic infectious contents comprised by a complex bacterial community was shown to be a potent activator of TLR4, determined by p38 MAPK and NF-kB signaling pathways, culminating in a high antigenicity against macrophages through the levels of IL-6 and IL10; all were signicantly affected by endotoxin levels and participated in the clinical ndings involved in endodontic disease. Further studies should be performed in order to evaluate the effect of root canal therapies on infection control of bacterial and fungi endodontic contents through the measurement of cytokines production. Also, p38 and NF-kB or the components of their pathways can be a therapeutic target for the development of new drugs against inammatory root canal diseases.
8. Stashenko P, Teles R, DSouza R. Periapical inammatory responses and their modulation. Crit Rev Oral Biol Med 1998;9:498521. 9. Garcia de Aquino S, Manzolli Leite FR, Stach-Machado DR, et al. Signaling pathways associated with the expression of inammatory mediators activated during the 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 human periodontal disease tissue. J Oral Pathol Med 1994;23:34753. 11. Socransky SS, Smith C, Martin L, et al. Checkerboard DNA-DNA hybridization. Biotechniques 1994;17:78892. 12. Oliveira LD, Carvalho CA, Carvalho AS, et al. Efcacy of endodontic treatment for endotoxin reduction in primarily infected root canals and evaluation of cytotoxic effects. J Endod 2012;38:10537. 13. Jacinto RC, Gomes BP, Ferraz CC, et al. Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with periapical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria. Oral Microbiol Immunol 2003;18:28592. 14. Vianna ME, Horz HP, Conrads G, et al. Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture. Oral Microbiol Immunol 2008;23:28290. 15. Poltorak A, Smirnova I, He X, et al. Genetic and physical mapping of the Lps locus: identication of the toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol Dis 1998;24:34055. 16. Hoshino K, Takeuchi O, Kawai T, et al. Cutting edge: Toll-like receptor 4 (TLR4)decient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:374952. 17. Riva M, Kallberg E, Bjork P, et al. Induction of nuclear factor-kappaB responses by the 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 TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS. Biochem Biophys Res Commun 2012;420:7627. 19. Sassone LM, Fidel RA, Faveri M, et al. A microbiological prole of unexposed and exposed pulp space of primary endodontic infections by checkerboard DNA-DNA hybridization. J Endod 2012;38:88993. 20. Moraes SR, Siqueira JF Jr, Colombo AP, et al. Comparison of the effectiveness of bacterial culture, 16S rDNA directed polymerase chain reaction, and checkerboard DNA-dNA hybridization for detection of Fusobacterium nucleatum in endodontic infections. J Endod 2002;28:869. 21. Martinho FC, Chiesa WM, Leite FR, et al. Antigenicity of primary endodontic infection 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 and TLR4 for cytokine production in human primary monocytes and macrophages. Cell Microbiol 2008;10:205866. 23. Gomes BP, Endo MS, Martinho FC. Comparison of endotoxin levels found in primary and secondary endodontic infections. J Endod 2012;38:10826. 24. Fong Y, Moldawer LL, Marano M, et al. Endotoxemia elicits increased circulating beta 2-IFN/IL-6 in man. J Immunol 1989;142:23214. 25. Huang GT, Do M, Wingard M, et al. Effect of interleukin-6 deciency on the formation of periapical lesions after pulp exposure in mice. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:838. 26. Tinsley JH, South S, Chiasson VL, et al. Interleukin-10 reduces inammation, endothelial dysfunction, and blood pressure in hypertensive pregnant rats. Am J Physiol Regul Integr Comp Physiol 2010;298:R7139. 27. Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal 2000;12:113. 28. ONeill LA. The interleukin-1 receptor/Toll-like receptor superfamily: 10 years of progress. Immunol Rev 2008;226:108. 29. Albiger B, Dahlberg S, Henriques-Normark B, et al. Role of the innate immune system 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 inhibits the activity but not the activation of p38 mitogen-activated protein kinase. Biochem Biophys Res Commun 1999;263:82531.

Acknowledgments
The authors would like to thank Ana Regina de Oliveira Polay for her technical assistance. They also are thankful to Lonza for the Kinetic-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 conicts of interest.

References
1. 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 macrophage 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 infected dental root canals. Oral Microbiol Immunol 2004;19:716. 5. Rocas IN, Siqueira JF Jr. Identication of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach. J Endod 2010;36:4552. 6. Schein B, Schilder H. Endotoxin content in endodontically involved teeth. J Endod 1975;1:1921. 7. Martinho FC, Chiesa WM, Leite FR, et al. Correlation between clinical/radiographic features and inammatory cytokine networks produced by macrophages stimulated with endodontic content. J Endod 2012;38:7405.

JOE Volume 40, Number 4, April 2014

Signaling Pathways Activation by Endodontic Infectious Contents

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