1http://dx.doi.org/10.20396/bjos.v19i0.8657039 Volume 19 2020 e207039 Original Article 1 Universidade Católica de Brasília - UCB, Centro de Análises Proteômicas e Bioquímicas. Post-Graduation Program in Biotechnology and Genomic Sciences, Brasília, Brazil. 2 Universidade Católica de Brasília - UCB, Pharmacy Department, Brasília, Distrito Federal, Brazil 3 Universidade Católica de Brasília - UCB, Dental Course, Brasília, Distrito Federal, Brazil 4 Universidade Federal de Mato Grosso do Sul - UFMS, Programa de Pós Graduação em Saúde e Desenvolvimento na Região Centro Oeste. Faculdade de Medicina, Campo Grande, Mato Grosso do Sul, Brazil. 5 Universidade Católica Dom Bosco - UCDB, S-Inova Biotech, Post-Graduation Program in Biotechnology, Campo Grande, Mato Grosso do Sul, Brazil 6 Universidade de Brasília - UNB, Post-Graduation Program in Molecular Phatology, Brasília, Distrito Federal, Brazil 7 Universidade de Brasília - UNB, Post-Graduation Program in Health Sciences, Brasília, Distrito Federal, Brazil Corresponding author: Taia Maria Berto Rezende Universidade Católica de Brasília - UCB Post-Graduation Program in Biotechnology and Genomic Sciences SGAN 916N – Av. W5 – Campus II – Modulo C, room C-221 Brasília-DF, Brazil Fone: + 55-61-981349001 Fax: + 55-61-3347-4797 e-mail: taiambr@gmail.com / taia@ucb.br Received: October 17, 2019 Accepted: February 24, 2020 Enterococcus faecalis and Staphylococcus aureus stimulate nitric oxide production in macrophages and fibroblasts in vitro Maurício Gonçalves da Costa Sousa1, Patrícia Diniz Xavier2, Stella Maris de Freitas Lima3, Jeeser Alves de Almeida4, Octávio Luiz Franco1,5,6, Taia Maria Berto Rezende1,3,7,* Aim: Nitric oxide (NO) is an important mediator related to damage of the pulp tissue and at the same time to regenerative pulp processes. However, it is not clear how common endodontic microorganisms can regulate this mediator. This study aimed to investigate NO production by macrophages and fibroblasts against Enterococcus faecalis- and Staphylococcus aureus-antigens. Methods: RAW 264.7 macrophages and L929 fibroblast cell lines were stimulated with different heat- killed (HK) antigen concentrations (105-108 colony forming units - CFU) from E. faecalis and S. aureus with or without interferon-gamma (IFN-γ). Cell viability by MTT colorimetric assay and NO production from the culture supernatants were evaluated after 72 h. Results: Data here reported demonstrated that none of the antigen concentrations decreased cell viability in macrophages and fibroblasts. The presence of HK-S. aureus and HK-E. faecalis antigen- stimulated NO production with or without IFN-γ on RAW 264.7. The HK-S. aureus antigen stimulated NO production in L929 fibroblasts with or without IFN-γ, and the highest concentration of HK-E. faecalis with IFN-γ also stimulated NO production by these cells. Conclusion: The amount of NO produced by macrophages and fibroblasts may be involved in the concentration and type of prevalent endodontic microorganisms, generating new answers for the understanding of pulpal revascularization/regeneration processes. Keywords: Enterococcus faecalis. Staphylococcus aureus. Fibroblasts. Macrophages. Nitric oxide. 2 Sousa et al. Introduction Regenerative endodontic therapies have become widespread, especially in imma- ture teeth with open apex1. Difficulties during root canal instrumentation and disad- vantages of the conventional apexification technique motivate pulp revasculariza- tion therapy. Briefly, this process consists of accessing the root canal system (RCS), irrigating it and subsequently using an intracanal medication aiming to remove the largest number of microorganisms in the RCS. This aseptic environment for blood clot formation is essential for the new tissue formation1,2. The success of regenera- tive therapies will depend on three important factors, namely the presence of stem cells, growth factors and scaffold3. Several mesenchymal stem cells, originating from the apical papilla (SCAP), dental pulp stem cells (DPSCs) or from exfoliated deciduous teeth (SHED) can contribute to pulp tissue regeneration4,5. However, the biomarkers needed for tissue reconstruction are still unknown. In addition, many clinical studies have reported the formation of fibroblast-rich scar tissue, without free nerve endings6. Until now, the role of other cells such as macrophages and fibroblasts in this process is unclear7. Moreover, the presence of some Gram-positive bacterial species such as Enterococcus faecalis has been found in revascularized tissues, triggering the production of mediators and pro-inflammatory cytokines, which may hamper tissue repair8. Among all the mediators produced by macrophages and fibroblasts, nitric oxide (NO) can act both in the elimination of invading agents and in the formation or destruction of tissues9. In this way, in a previous study, NO was upregulated in the presence of E. faecalis in vitro10. In relation to the formation of new tissues, low concentrations of NO may stimulate new vessel formation during pulpal regeneration / revasculariza- tion processes11. However, on the other hand, high concentrations can cause pulp tis- sue damage and particularly hinder new tissue formation9. This happens because NO may be associated with odontoblast differentiation and the production of enzymes and proteins related with bone and dentin formation, including alkaline phosphatase and calcitonin12,13. Therefore, considering the difficulty of promoting an aseptic environment, the pur- pose of this study was to evaluate in vitro cell viability and the production of NO in two cell lines. These cells were stimulated with different concentrations of heat-killed antigens from prevalent endodontic bacterial E. faecalis and S. aureus, mimicking the environment related to pulp revascularization. Materials and methods RAW 256.7 and L929 fibroblast cultures RAW 264.7 osteoclast precursor monocyte cells (CR108, Rio de Janeiro Cell Bank, Rio de Janeiro, Brazil) were cultured at 1x105 cells per well in 96-well culture plates (TPP, USA). L929 fibroblasts (ATCC 929) were cultivated at 1x105 per well in 96-well culture plates (TPP, USA). Both cells were cultured in Dulbecco modified Eagle medium (Gibco, USA) supplemented with 10 % fetal bovine serum (Gibco, USA), 1 % 3 Sousa et al. penicillin / streptomycin (1000 U.mL-1) (Invitrogen, Grand Island, NY), 1 % nonessen- tial amino acids (Invitrogen), 1% L-glutamine and 0.1% gentamicin (Invitrogen)14,15. These cell cultures were stimulated in vitro with different concentrations of heat- killed antigens (HK) from E. faecalis (ATCC 19433) and S. aureus (ATCC 25923) with or without recombinant (r) IFN-γ (10 U per well, Peprotech, USA), mimetizing the endodontic environment in the necrosis of incomplete rhizogenesis processes. As a control, both cells were also stimulated with lipopolysaccharide (LPS) (3 µg.ml-1, Sigma-Aldrich, USA)16. Cell viability assay and NO production were assessed after 72 h of incubation. HK antigen preparations Experimental groups determined for cytotoxicity and NO production analyses were stimulated with HK-E. faecalis and -S. aureus. Heat-killed antigens were prepared, as previously described10. Briefly, colonies were grown in Luria Bertani agar (LB; Kasvi, pH 7.3; USA) and subsequently resuspended in sterile phosphate-buffered saline solution, followed by their quantification by optical density. Then, they were heated at 121 °C, for 50 min10. Different concentrations of antigens from both microorganisms (105-108 colony-forming units/well) were tested. Death of microorganisms was confirmed by the absence of colonies, after 24 hours of incubation in Luria Bertani agar (LB; Kasvi, pH 7.3; USA). Optical microscopy images (inverted microscope Axiovert 40 CFL, USA; objective 20x) were obtained after 72h of incubation, from the experimental groups stimulated with rIFN-γ, LPS, and rIFN-γ, HK-S. aureus (106 CFUs) with or without rIFN-γ and HK-E. faecalis (106 CFUs) with or without rIFN-γ. Viability assay Cell viability assays were performed on both cells with antigen stimulus after a period of 72 h incubation at 37 °C, 5 % CO2 and 95 % humidity, with 3-[4,5-dimeth- ylthiazol-2-yl]-2,5-diphenylte-trazolium (MTT) bromide (0.25 mg.mL-1). The absor- bance was measured at 595 nm in a microplate spectrophotometer (Bio-Tek, Win- ooski, VT). The results were compared to a positive control (unstimulated cells) and negative controls (cell culture in lysis buffer solution - 10 mmol.L-1 Tris, pH 7.4, 1 mmol.L-1 EDTA and 0.1% Triton X-100) and plotted as mean±standard error of absorbance16. Nitric oxide production NO levels in culture supernatant from both stimulated cell lines were determined by Griess reaction. After 72h of incubation, the supernatant was mixed with an equal vol- ume of Griess reagent (1 % sulfanilamide and 0.1 % naphtylethylene in 2.5 % ortho-phos- phoric acid; Sigma-Aldrich, GB, Brazil). The absorbance was measured by a microplate spectrophotometer (Bio-Tek, Winooski, VT) at 490 nm. The nitrite concentration was determined according to a standard curve (0–200 mmol.L-1 sodium nitrite)17. Statistical Analysis All experiments were carried out in technical and biological triplicates. Statistical anal- yses were performed by Kolmogorov-Smirnov test followed by one-way analysis of 4 Sousa et al. variance (ANOVA) and Bonferroni post hoc by using GraphPad Prism 6 (GraphPad Software, San Diego, CA); p<0.05 was considered statistically significant. Results Cellular viability related to microbial antigen and controls First, the cell viability of both chosen cell lines was evaluated, in the presence of HK-E. faecalis or HK-S. aureus. In this context, antigen concentration may be deter- minant for cell viability. Thus, RAW 264.7 stimulated with different concentrations of S. aureus did not diminish cell viability and the LPS-stimulated group induced cell proliferation (Fig. 1A). Otherwise, the addition of rIFN-γ and 108 CFU.mL-1 of S. aureus upregulated cell proliferation (Fig. 1C). Regarding E. faecalis, this antigen did not reduce cell viability and, at 108 CFU.mL-1, it was also able to increase cell viability (Fig. 1B). Furthermore, the LPS-control group induced cell proliferation, while the presence of rIFN-γ did not represent an additional stimulus to alter cell viability (Fig. 1C and 1D). In order to relate our viability results with the morphological charac- teristics presented by these cells, the optical microscopy images (Fig. 1A-L) were observed, and these showed that after three days’ incubation, the groups contain- ing HK-S. aureus (Fig. 1I and 1J) and HK-E. faecalis (Fig. 1K and 1L) antigens with or without rIFN-γ significantly altered the cellular morphology of RAW 264.7, when compared to the control group (Fig. 1E). Regarding L929 fibroblasts cell viability, none of the concentrations studied was cyto- toxic. Therefore, neither L929 fibroblasts stimulated with different concentrations of S. aureus, nor LPS, diminished cell viability (Fig. 2A). Besides, the highest concentration of this HK upregulated cell proliferation (Fig. 2C). A similar relationship was observed when the rIFN-γ was added to HK-S. aureus. These stimuli did not reduce cell viability and, at 106 CFU.mL-1, they also stimulated cell proliferation (Fig. 2C). HK-E. faecalis did not reduce cell viability, and at 106, 107 and 108 CFU.mL-1, it again increased cell viabil- ity (Fig. 2B). The rIFN-γ stimulus was not able to alter cell viability in any of the tested groups (Fig. 2D). Concerning the morphological alterations by optical microscopy, the images demonstrated the structural differences under stress that both cells may present in the presence of both tested antigens. Thus, the optical microscopy images (Fig. 1A-L) showed that after three days’ incubation, groups containing LPS (Fig. 1G), HK-S. aureus (Fig. 1I and 1J) and HK-E. faecalis (Fig. 1K and 1L) antigens with or with- out rIFN-γ significantly altered cellular morphology of L929, when compared to the control group (Fig. 1E). NO production related to microbial antigen and controls The evaluation of NO production was performed after 72h in both cells studied. Assembling a system involving both the different antigens and the presence of the rIFN-γ recombinant, it was possible to mimic an in vitro infection. In this way, in RAW 264.7 cultures, the presence of LPS and different concentrations of HK-S. aureus was able to upregulate the production of sodium nitrite, after 72 hours of incubation, except for the group containing 108 CFU.mL-1 (Fig. 3A). Therefore, the presence of IFN-γ increased nitrite production by these cells in different concentra- 5 Sousa et al. tions of S. aureus or LPS, except for the group stimulated by 108 CFU.mL-1 (Fig. 3C). Only the 108 CFU.mL-1 of E. faecalis stimulus was able to induce the production of sodium nitrite (Fig. 3B). The addition of rIFN-γ led to an increase in sodium nitrite production, except for the group containing 108 CFU.mL-1 of E. faecalis (Fig. 3D). Regarding the L929 cultures, these cells may represent the most abundant cells on the pulp tissue: fibroblasts. Then, the presence of LPS or different HK-S. aureus Figure 1. RAW 264.7 cell viability. Graphs represent cell cultures with different concentrations (105-108 CFU.mL-1) of HK-S. aureus antigen (A) with rIFN-γ (C) or HK-E. faecalis antigen (B) with rIFN-γ (D), after 72h of cell incubation. Bars represent mean and standard error of cellular absorbance (595 nm) carried out in technical and biological triplicates. Statistical differences by one-way ANOVA test and Bonferroni post hoc were represented by * (p < 0.05), ** (p < 0.01) and *** (p< 0.001). Optical microscopy (20x) shows the initial (day 1) and final (day 3) cell morphology aspects (E-K) of RAW 264.7 stimulated with rIFN-γ (F), LPS (G), LPS and rIFN-γ (H), HK-S. aureus 106 CFUs without (I) or with rIFN-γ (J), HK-E. faecalis 106 CFUs without (K) or with rIFN-γ (L) compared to the cell control group (E). +IFN-γ +LPS +IFN-γ +LPS +HK S. aureus +HK S. aureus +IFN-γ +HK E. faecalis +HK E. faecalis +IFN-γ D ay 1 D ay 3 (E) (F) (G) (K)(J)(I)(H) (L)Control (A) (B) (C) (D) A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 ** ** ** *** ** ** HK – S. aureus HK – E. faecalis HK – S. aureus + rIFN-γ HK – E. faecalis + rIFN-γ RAW LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 RAW LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 RAW LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 RAW LPS + IFN-γ 105 106 107 108 user Highlight conferimos as provas anteriores e não encontramos a indicação da correção para (μM) nas figuras 1 e 2. devemos corrigir? 6 Sousa et al. concentrations were capable of stimulating the production of sodium nitrite, with or without rIFN-γ (Fig. 4A and 4C). Only the highest concentration of HK-E. faecalis was able to stimulate the production of sodium nitrite without rIFN-γ (Fig. 4B). Nevertheless, when the rIFN-γ was added to all groups, sodium nitrite production was upregulated (Fig. 4D). Figure 2. L929 cell viability. Graphs represent cell cultures with different concentrations (105-108 CFU. mL-1) of HK-S. aureus antigen (A) with rIFN-γ (C) or HK-E. faecalis antigen (B) with rIFN-γ (D), after 72h of cell incubation. Bars represent mean and standard error of cellular absorbance (595 nm) carried out in technical and biological triplicates. Statistical differences by one-way ANOVA test and Bonferroni post hoc were represented by * (p < 0.05). Optical microscopy (20x) shows the initial (day 1) and final (day 3) cell morphology aspects (E-K) of L929 stimulated with rIFN-γ (F), LPS (G), LPS and rIFN-γ (H), HK-S. aureus 106 CFUs without (I) or with rIFN-γ (J), HK-E. faecalis 106 CFUs without (K) or with rIFN-γ (L) compared to the cell control group (E). A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 * * * * * HK – S. aureus HK – E. faecalis HK – S. aureus + rIFN-γ HK – E. faecalis + rIFN-γ L929 LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 L929 LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 L929 LPS + IFN-γ 105 106 107 108 A bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 L929 LPS + IFN-γ 105 106 107 108 +IFN-γ +LPS +IFN-γ +LPS +HK S. aureus +HK S. aureus +IFN-γ +HK E. faecalis +HK E. faecalis +IFN-γ D ay 1 D ay 3 (E) (F) (G) (K)(J)(I)(H) (L)Control (A) (B) (C) (D) 7 Sousa et al. Figure 3. NO production by RAW 264.7 cells. Graphs represent values of sodium nitrite with different concentrations (105-108 CFU.mL-1) of HK-S. aureus antigen (A) with rIFN-γ (C) or HK-E. faecalis antigen (B) with rIFN-γ (D), after 72h of cell incubation. Bars represent mean and standard error of sodium nitrite production in µM carried out in technical and biologic triplicates. Statistical differences by one-way ANOVA test and Bonferroni post hoc were represented by *** (p< 0.001) and **** (p < 0.0001). - - + - - - - - *** *** **** **** - - - -- - - + **** **** - - - - - - - + + + ++ - + **** **** **** **** - - --- - - - ++ + + + + **** **** *** **** 105 106 107 108 N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 HK – E. faecalis N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 HK – E. faecalis 105 106 107 108 N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 rIFN-g (10U per well) HK – E. faecalis 105 106 107 108 0 1 2 3 LPS 3 µg.mL-1 rIFN-g (10U per well) HK – E. faecalis 105 106 107 108 (A) (B) (C) (D) N itr ite (µ M ) Figure 4. NO production by L929 fibroblasts. Graphs represent values of sodium nitrite with different concentrations (105-108 CFU.mL-1) of HK-S. aureus antigen (A) with rIFN-γ (C) or HK-E. faecalis antigen (B) with rIFN-γ (D), after 72h of cell incubation. Bars represent mean and standard error of sodium nitrite production in µM carried out in technical and biologic triplicates. Statistical differences by one-way ANOVA test and Bonferroni post hoc (p<0.05) were represented by * (p < 0.05), ** (p < 0.01) *** (p< 0.001) and **** (p < 0.0001). N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 HK – E. faecalis ---- -- - 105 106 107 108 + **** **** * ** **** N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 HK – E. faecalis -- -- ----+ 105 106 107 108 *** * N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 rIFN-g (10U per well) HK – E. faecalis - - - ---- + ++++ 105 106 107 108- + **** * ** ** * N itr ite (µ M ) 0 1 2 3 LPS 3 µg.mL-1 rIFN-g (10U per well) HK – E. faecalis + ------ ---- -- - 105 106 107 108 ++ ++ + **** * (A) (B) (C) (D) 8 Sousa et al. Discussion Regenerative therapies may contribute to endodontic treatment in immature teeth with open-apex3,18. However, both the absence of microorganisms and the pres- ence of mediators and growth factors are essential for the construction of new pulp tissue18. Thus, the polymicrobial infected root canal system is composed of Gram-positive and Gram-negative bacteria. Among the microorganisms, E. faeca- lis is prevalent in infected immature permanent teeth19. Moreover, this microorgan- ism is associated with different forms of periradicular disease, including primary endodontic infections as well as persistent periapical lesions20. In the category of primary endodontic infections, E. faecalis is present in 40% of them20. S. aureus might be another bacterium found in pulpitis and may have quorum-sensing as its main mechanism of virulence21. This factor contributes to the control of the patho- genesis of this microorganism, which is involved with the density that occurs through cellular communications22. The presence of microorganisms may inhibit the development of new tissue, mod- ifying the normal function of these cells23. In an in vitro study, LPS from Pseudo- monas aeruginosa did not reduce cell viability, but reduced the ability of periodon- tal ligament stem cells to differentiate into osteoblasts; in addition, it upregulated the production of proinflammatory cytokines such as IL-1β, IL-6, and IL-8 by these cells23. In the same way, the activation of immune system cells, metalloproteinase, reactive oxygen species (ROS) and bacterial endotoxins, for instance LPS and lipo- teichoic acid (LTA), may compromise the development of loose connective tissue24. It has been described that the presence of S. aureus antigens downregulated the bone marrow stem cell and human fibroblast adhesion factors, by blocking TLR225. In addition, LTA from S. aureus walls was related with the production of NO in RAW 264.7 macrophages, via TLR226. The antigen-fighting process of resistant microorganisms is mostly associated with the first line of immune response, represented by cytokines and lysosomal enzyme-macrophage producers, related to tissue destruction27. However, fibroblasts are the main cells present in connective tissue, deploying a structural and repair role, including the release of tissue repair mediators28. Thus, for this study, RAW 264.7 macrophages and L929 fibroblasts were chosen. In this context, IFN-γ may be responsible for upregulating the class I and II major histocompatibility complexes and activating reduced nicotinamide adenine dinucleotide phosphate–dependent phagocyte oxidase and NO production in macrophages, besides exacerbating the response to the production of NO in fibroblasts29,30. In this study, the S. aureus and E. faecalis stimuli in RAW 264.7 macrophages were not able to decrease cell viability, even at higher tested concentrations. However, the HK antigens altered cell morphology at all tested concentrations. These results were also observed in a previous study, in which RAW 264.7 cells remained viable even at higher S. aureus antigen concentrations, after 48 h of incubation31. The presence of different concentrations of S. aureus and E. faecalis antigens also did not diminish the L929 fibroblast viability. This is the first study, according to our knowl- edge, that has evaluated the effects of Gram-positive bacteria on the L929 cell line. 9 Sousa et al. The presence of heat-killed Porphyromonas gingivalis in fibroblasts from periodontal ligament was not cytotoxic, after 48 h of incubation, even at the highest cells: bacteria proportion (1:100)32. The main events reported with the presence of antigens in the root canal systems are related to changes in the pattern of response and production of mediators by these cells10. Among these mediators, NO is a gaseous free radical produced by NO-synthase, by converting L-arginine to L-citrulline33. The action of inducible nitric oxide (NO2) on pulp tissue can contribute to the destruction of microorganisms, but at the same time, high concentrations (500 µM) were able to cause apopto- sis of macrophages and osteoblasts in an in vitro periapical lesion model34. The beneficial or malignant action of NO may be related to the levels of NO produced. Low concentrations of NO in pulp space can contribute to tissue formation and regeneration processes, since the formation of new vessels may be essential for the construction of new tissue11. And because it is lipophilic, NO can easily be per- meable to biological membranes, causing vasodilation35. In addition, NO (100 µM) may upregulate the vascular endothelial growth factor (VEGF), which is essential for angiogenesis36. As the pulp tissue have a higher concentration of blood ves- sels, the synthesis of this free radical becomes essential in the support and estab- lishment of their physiology11. An in vitro study demonstrated an increase in NO synthase expression in pulp cells derived from immature permanent teeth when compared to third molar pulp cells37. This study showed that both stimuli (S. aureus and E. faecalis) were able to induce the production of NO in RAW macrophage 264.7, based on a standard curve of sodium nitrite. NO production in macrophages in the presence of S. aureus stimuli seems to be dose dependent. However, at the highest concentration the abundance of this spe- cific mediator was not improved. In this context, macrophages are the first defense line and, when in contact with antigens, are specialized in producing inducible NO38. Macrophage polarization (M1) may perpetuate an inflammatory response, whereas a macrophage response (M2) may contribute to the formation of new tissues. An in vitro study associated the autocrine action of NO on LPS-stimulated macrophages with the polarization in profile M139. Virulence factors such as LTA from Gram-positive bacteria, peptide glycol and adhesion factors are generally associated with the induction of NO synthase in macrophages32,40. Here, the higher antigen concentrations, in the presence of IFN- γ, did not stimulate NO production in RAW 264.7 cells. In situations of high antigen concentrations, the immune cells can lose their response pattern and may act unresponsively due to the mechanisms of immune regulation mediated by regula- tory T lymphocytes39,41. The presence of S. aureus antigens stimulated NO production at all tested concentra- tions, with or without IFN-γ in L929 fibroblasts. In the presence of E. faecalis, the high- est concentration of antigens was significantly important in inducing NO production. Fibroblasts are known to have an important role in tissue repair; however, they also respond to the antigen by producing IL-6, MCS-F, TGF-β, and NO42,43. Until then, the classical stimuli studied for the evaluation of NO production in L929 are IFN-γ, LPS or both30. In this way, NO production in human pulp fibroblasts in response to heat-killed 10 Sousa et al. antigens from E. faecalis may increase alkaline phosphatase production in fibroblasts and, consequently, pulp calcification44. In addition, the production of NO and other proinflammatory cytokines in fibroblasts may favor the expression of OPG in these cells and consequently the formation of calcified pulp tissue45. Besides, fibroblasts may be susceptible to NO. An in vitro study demonstrated that 3 mmol.L-1 of NO were responsible for the apoptosis of gingival fibroblasts. This action was associated with the c-Jun N-terminal kinase signaling pathway46. In conclusion, NO production by RAW 264.7 monocytes and L929 fibroblasts against the pathogens presented in this study may contribute to the understanding of how microorganisms prevalent in the root canal system lead to a pro-inflammatory response, increasing NO. This is an initial study and in view of the real role of this mediator, new studies with human cells must be carried out to establish its action both in the elimination of microorganisms and in the formation of new tissues during pulp revascularization/regeneration processes. Acknowledgements This study was supported by the Universidade Católica de Brasília (UCB), Conselho Nacional de Desenvolvimento Tecnológico (CNPq), Coordenação de Aperfeiçoa- mento de Pessoal de Nível Superior (CAPES), Fundação de Amparo do Distrito Federal (FAPDF) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT). The authors deny any conflicts of interest. References 1. Yang J, Yuan G, Chen Z. Pulp regeneration: current approaches and future challenges. Front Physiol. 2016 Mar 7;7:58. doi: 10.3389/fphys.2016.00058. eCollection 2016. 2. Galler, KM Clinical procedures for revitalization: current knowledge and considerations. Int Endod J. 2016 Oct;49(10):926-36. doi: 10.1111/iej.12606. 3. Dhillon H., Kaushik M, Sharma R. Regenerative endodontics - Creating new horizons. J Biomed Mater Res B Appl Biomater. 2016 May;104(4):676-85. doi: 10.1002/jbm.b.33587. 4. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. 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