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Phenotypic Analysis of Immunocompetent Cells in Healthy Human Dental Pulp
Clinical Research
Phenotypic Analysis of Immunocompetent Cells in Healthy Human Dental Pulp Alexis Gaudin, DDS,*† Emmanuelle Renard, DDS,† Marcello Hill, MD, PhD,† Laurence Bouchet-Delbos, MEng,† Geraldine Bienvenu-Louvet, PhD,* Jean-Christophe Farges, DDS, PhD,‡ Maria-Cristina Cuturi, MD, PhD,† and Brigitte Alliot-Licht, DDS, PhD*† Abstract Introduction: Like other tissues in the body, the human dental pulp is equipped with a network of immune cells that can be mobilized against pathogens when they invade the tooth. Very little data, mostly obtained with classic histologic methods, have reported their quantities and relative percentages. The objective of this study was to characterize and precisely quantify immunocompetent cells in healthy human dental pulp by using fluorescence-activated cell sorting, together with identifying specific cell subsets in the leukocyte (CD45+) cells. Methods: Healthy human third molars were collected from 42 young patients. Dental pulps were separated from the hard tissues and prepared for flow cytometry or immunostaining analyses. Results: CD45+ cells represented 0.94% 0.65% of cells obtained from the enzymatic digestion of whole dental pulps (n = 34). CD16+CD14+ granulocytes/neutrophils (50.01% 9.08%, n = 7) were found to represent the major subpopulation in CD45+ cells followed by CD3+ T lymphocytes (32.58% 11%, n = 17), CD14+ monocytes (8.93% 5.8%, n = 7), and HLADRhigh Lin1- dendritic cells (4.51% 1.12%, n = 7). Minor subpopulations included CD3 CD56+ natural killer cells (2.63% 1.15%, n = 7) and CD19+ B lymphocytes (1.65% 0.89%, n = 17). We further identified cells harboring a phenotype compatible with Foxp3/CD25-expressing regulatory T lymphocytes (CD45+CD3+CD4+CD127low). Fluorescence-activated cell sorting analysis and confocal microscopy also revealed expression of HO-1 in HLA-DR+ cells. Conclusions: For the first time, this study identifies and precisely quantifies the relative proportion of immunocompetent cells potentially involved in tissue homeostasis of healthy human dental pulp. (J Endod 2015;-:1–7)
Key Words Fluorescence-activated cell sorting, healthy dental pulp, immune cells, immunosurveillance
H
uman dental pulp is a highly dynamic tissue equipped with a network of resident immunocompetent cells that are believed to play a major role in the maintenance of tissue homeostasis. Injuries such as dental caries, trauma, operative procedures, and periodontal diseases are susceptible to break this balance. Dental caries result from a complex process in which the composition of the oral bacterial biofilm evolves as the penetration of microorganisms through the enamel, dentin, and pulp occurs (1). Bacteria and their toxins present in dentinal tubules trigger several important cellular and molecular changes within the pulpal tissues. Like any other injured tissue in the body, dental pulp is able to mount innate adaptive immune responses intended to fight infection (2, 3). During bacterial penetration through the dentin, inflammatory mediators can be released by acidic degradation of the carious dentin and produced by resident pulp cells such as odontoblasts, pulpal fibroblasts, stem cells, endothelial cells, and/or tissue-resident immune cells (1, 4). Because of their peripheral localization, odontoblasts are considered the first line of cell defense of the pulp tissue. They express a variety of pathogen recognition receptors and chemokines and have been proposed to trigger the initial inflammatory and immune pulp response in cooperation with antigen-presenting dendritic cells (DCs) (5, 6). DCs, macrophages, T lymphocytes, natural killer (NK) cells, and B lymphocytes have been identified in inflamed human dental pulp based on histologic analysis (7, 8). Immune cells like DCs and T lymphocytes are also known to be resident in healthy pulp where they play a role in immunosurveillance. Immunosurveillance is a term used to describe the processes by which cells of the immune system look for and recognize foreign pathogens, such as bacteria and viruses, in the body. Immunosurveillance is well documented for skin, mucosa, lung, blood, and brain tissues. To our knowledge, precise characterization and quantification of immunocompetent cells in healthy human pulp have not been reported (2, 3). Fluorescence-activated cell sorting (FACS) is widely used to identify and characterize specific cell subsets in cellular suspensions obtained from tissues and is a great help in the determination of cell percentages in the tissues. Thus, FACS gives the opportunity to identify small cell populations with a set of different markers within the whole dental pulp tissue. Precise quantification, relative repartition among the leucocyte population, and detection of specific immunoregulatory cells will lead to a better understanding of the pulp initial capacity to mount efficient immune responses and regulate them. This knowledge is important to better
From the *Faculty of Odontology, University of Nantes, Nantes, France; †INSERM, Institut de Transplantation et de Recherche en Transplantation, Nantes, France; and ‡Laboratory of Tissue Biology and Therapeutic Engineering, University of Lyon, Lyon, France. Address requests for reprints to Dr Alexis Gaudin, Faculte de Chirurgie Dentaire, 1 Place Alexis Ricordeau, 44042 Nantes, Cedex 1, France. E-mail address: alexis. [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.01.005
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Phenotypic Analysis of Immunocompetent Cells
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Clinical Research understand the physiopathology of the pulp during bacterial aggression. Using FACS analysis, our study aimed to characterize and precisely quantify immunocompetent cells and identify specific subsets among the leukocytes (CD45+ cells) present in healthy human dental pulp. Furthermore, the detection of subsets involved in immunoregulation should lead to the discovery of targets for future therapeutic strategies.
Materials and Methods Collection of Pulp Samples Tissue samples were obtained from 42 healthy patients between the ages of 11 and 25 years who were elected to have extractions of healthy third molars under general anesthesia for orthodontic reasons. Teeth were collected with patients’ (or the parents of patients) informed consent. The surgeries were performed at the Clinic of Stomatology of Nantes under approved guidelines. Impacted or embedded teeth that were cut during removal were excluded. Freshly extracted molars were taken directly from the surgical site and placed into sterile phosphate buffered saline (PBS) (pH = 7.4). Attached soft tissue was removed from the root surface by sterile scalpel blades. They were then cracked open, and the pulp tissue was gently removed with cotton pliers and placed in a Petri dish containing 1 mL PBS. Tissue Preparation Pulp tissues were minced into small pieces (0.5 mm3) using sterile scalpel blades, transferred into a 50-mL sterile tube, and centrifuged at 1500 rpm for 10 minutes. The supernatant was removed, and the tissue pellet was digested in 5 mL collagenase D (Roche, Meylan, France) and then incubated at 37 for 45 minutes. The pulp tissue was completely dissociated in 30 to 45 minutes. EDTA (10 mmol/L) was added for the last 5 minutes to stop the enzymatic digestion. The cell suspension was washed twice with 50 mL sterile PBS, passed through a 100-mm nylon mesh filter, and resuspended in 2 mL PBS supplemented with 2 mmol/L EDTA and 2% fetal calf serum (FCS). We routinely obtained 1 to 7 million cells per patient, which were subsequently used for antibody staining. Flow Cytometry For cell surface staining, suspensions of single pulp cells were distributed into 96-well V-bottomed plates, washed, and incubated in PBS/2% FCS/0.2% azide. According to the manufacturer’s recommendations, cells were incubated on ice for 30 minutes with anti-CD45 PECy7 (clone HI-30, mouse immunoglobulin [Ig] G1) and monoclonal conjugated antibodies (mAbs [Table 1]) or isotype controls. The following combinations were used to identify pulp immunocompetent cells: B lymphocytes: CD45/CD19; T lymphocytes: CD45/CD3/CD4/
CD8; NK cells: CD45/CD3/CD56; DCs: CD45/HLA-DR/Lin1; and monocytes, granulocytes, and macrophages: CD45/CD14/CD16. Single-cell suspensions were washed twice with PBS/0.2% FCS/0.1% azide before fixation with 2% paraformaldehyde and then maintained in the dark at 2 C–8 C until further use. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of other patients by Ficoll-Hypaque density gradient centrifugation (Amersham, Les Ulis, France) in order to be used as positive controls. Flow cytometry analysis was performed by using the BD FACS Canto Flow Cytometer (Becton Dickinson, San Jose, CA). Results were analyzed using FlowJo analysis software (Tree Star, Ashland, OR). Primary gates based on physical parameters (forward and side light scatter) and fixable viability dye eFluor450 (eBioscience, Paris, France) were used in all experiments to exclude dead cells or debris. For intracellular staining, cells were distributed into 96-well Vbottomed plates, washed, and incubated in PBS 0.5% saponin for 20 minutes on ice. After 3 washes in PBS 0.1% saponin, cells were incubated for 30 minutes on ice according to the manufacturer’s recommendations with anti-CD45 PE-Cy7 (clone HI-30, mouse IgG1) and mAbs (Table 1) or isotype controls. The following mAb combinations were used: DCs–heme oxygenase-1 (HO-1): CD45/HLA-DR, regulatory T lymphocytes (Tregs) CD45/CD3/CD4/CD127/Foxp3, and CD45/CD3/ CD127/CD4/CD25. After 2 washes with PBS/0.2% FCS/0.1% saponin and 1 wash with PBS/2% FCS/0.2% azide, cells were fixed with 2% paraformaldehyde and analyzed as previously described.
Immunohistology and Confocal Image Analysis For immunohistology, third molar dental pulps from healthy donors were frozen and cryosectioned (10 mm). Tissue sections were fixed with acetone and saturated using PBS/4% bovine serum albumin/10% goat serum. Slides were then permeabilized with 0.5% saponin and stained with anti–HO-1 primary mAb (Stressgen, San Diego, CA). Slides were then incubated with biotinylated conjugated mAb diluted at 1/500. A second incubation was performed with streptavidin 568 conjugated biot mAb diluted 1/1000. Cells were also stained with anti-HLA-DR FITC mAb (BD Biosciences, Le Pont de Claix, France). Matching isotype control mouse antibodies were included. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Slides were mounted in ProLong AntiFade reagent (Molecular Probes, Saint-Aubin, France) and analyzed with a Leica confocal microscope (Leica Microsystems, Heidelberg, Germany) and Leica TCS NT software.
Results For the first time, we propose a precise quantification of leukocytes within the healthy human dental pup. Cellular suspensions were obtained
TABLE 1. Monoclonal Antibodies Used in This Study Antibodies CD45 CD19 CD3 CD4 CD8 HLA-DR Lin1 CD14 CD16 HO-1 CD127 CD25 Foxp3
Fluorochrome
Isotype
Clone
Manufacturer
PE-Cy7 PE APC-Cy7 PerCP-Cy5.5 FITC V500 FITC PE FITC FITC PE FITC APC
Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG2a Mouse IgG2b Mouse IgG2a Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Rat Ig2a
HI-30 HIB19 SK7 L200 HIT8a G46-6 NCAM16.2 M5E2 3G8 HO-1-1 HIL-7R-m21 M-A251 PCH101
BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences Abcam, Cambridge, UK BD Biosciences BD Biosciences eBioscience
Ig, immunoglobulin.
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Figure 1. Quantification of leukocytes (CD45+) in healthy human dental pulp. (A) The gating strategy used for the elimination of dead cells, debris, and doublets. Representative profiles of 1 experiment are shown. (B) Flow cytometry analysis showing cells stained with CD45 antibody or the appropriate fluorochromeconjugated control IgG. (C) The percentage of CD45+ represented 0.94% 0.65% of cells obtained after enzymatic digestion of the whole healthy human dental pulp (n = 34).
after enzymatic digestion, and all the results in this study were based on the same gating strategy to generate comparable data (Fig. 1A). Debris, nonviable cells, and doublets were excluded. Viable cells were found to represent 84.35% 5.8% of the whole pulp cells (n = 34) (data not shown). The percentage of leukocytes (CD45-expressing cells) was 0.94% 0.65% of the total viable pulp cells (Fig. 1B). Results were similar for the 34 different donors tested (Fig. 1C). We then investigated the expression of the phenotypic markers CD19, CD3, CD56, HLA-DR CD14, and CD16 by CD45+ leukocytes to determine the relative proportions of the major subsets of immunocompetent cells (Fig. 2A and B). We found that CD16+CD14+ granulocytes/ neutrophils represent the major subpopulation among CD45 + cells (50.01% 9.08%, n = 7) followed by CD3+ T cells (32.58% 11%, n = 17), CD14+ monocytes (8.93% 5.8%, n = 7), and HLA-DRhigh Lin1- DCs (4.51% 1.12%, n = 7). Minor populations included CD3 CD56+ NK cells (2.63% 1.15%, n = 7) and CD19+ B lymphocytes (1.65% 0.89%, n = 17). This repartition was homogenous among the donors tested. Lymphocytes (CD45+CD3+) are a major component of key immunocompetent cells. The lymphocyte population is mainly composed of CD4+ helper (or regulatory) and CD8+ cytotoxic TCRab+ T cells. The CD45+CD3+ lymphocyte population can also include NK T cells, mucosa-associated invariant T cells, or TCRgd+ T cells. Helper and cytotoxic lymphocytes were clearly identified by our flow cytometry analysis (Fig. 3A). Our data suggested that the helper T lymphocyte population (CD45+CD3+CD4+) was smaller than the cytotoxic T lymphocyte population (CD45+CD3+CD8+) (11.18% 0.5% vs 21.6% 2.8%, respectively), resulting in a CD4/CD8 ratio of 0.51. JOE — Volume -, Number -, - 2015
This ratio is different than the ratio obtained on PBMCs (Fig. 3B). Furthermore, we identified cells harboring a phenotype compatible with regulatory T lymphocytes (CD45+CD3+CD4+CD127low) expressing Foxp3 and CD25 (Fig. 3C). We also quantified the proportion of NK T lymphocytes (CD45+CD3+CD56+) and found that these cells represented 2.45% 1.34% of the total CD45+ cells (data not shown). We finally examined whether DCs possess immunoregulatory components in healthy dental pulp. We found HLA-DR+ cells expressing the enzyme HO-1 (Fig. 4A). Confocal microscopy showed the cytoplasmic expression of HO-1 in the HLA-DR+ cells (Fig. 4B).
Discussion Peripheral organs, such as the skin, gastrointestinal tract, and lungs, are barriers against invading microorganisms. In the healthy state, these organs contain sentinels cells including DCs, macrophages, and T lymphocytes to counteract pathogen invasion upon barrier breach within these organs. Dental pulp also contains a variety of immunocompetent cells (9–11). However, a better understanding of this background is necessary to understand the steady state of the pulp before comparison with inflamed tissue. In the field of endodontic research, Mangkornkarn et al (12) were the first to use FACS to examine the cellular components of human dental pulp tissue. FACS is a highly sensitive tool for cell counting compared with immunolabeling of histologic sections (7, 8, 12–14). FACS analysis gives the opportunity to combine several antibodies and to use positive or negative selection methods to identify and quantify specific cell populations in a reliable way (15). In this study, we
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Figure 2. Relative repartition of key immunocompetent cells among the CD45 + population. (A) Flow cytometry histograms of specific markers (black histogram), CD19, CD3, CD56, HLA-DR, CD14, and CD16, expressed among the CD45+ cells versus isotype controls (gray histogram) of 1 representative patient. Numbers represent the percentage of positive cells. (B) The level of expression of the following phenotypic markers: CD19+ for B lymphocytes, CD3+ for T lymphocytes, CD3 CD56+ for NKs, HLA-DRhighlin1- for dendritic cells, CD14+ for monocytes and macrophages, and CD16+CD14 for others (granulocytes and neutrophils) among CD45+ cells. The pie chart summarizes the relative repartition of key immunocompetent cells among the CD45 + population.
wanted to characterize the distribution of the cell subtypes within the leukocyte population present in healthy human dental pulp. Leukocytes were found to represent less than 1% of the cell population obtained after enzymatic digestion of the whole pulp tissue. These cells function to detect pathogens, allergens, and other harmful environmental agents that penetrate into the body (16). In our experiments, teeth had never been in contact with pathogens because they were extracted before any contact with the oral cavity. The percentage of leukocytes is relatively high for healthy connective tissue (16). This indicates that dental pulp contains the proper armamentarium to detect future pathogens. In our study, we found that CD8+ T lymphocytes (21.6% 2.8%) outnumbered CD4+ T lymphocytes (11.18% 0.5%) in healthy conditions. T lymphocytes are recognized as normal resident cells of human and rat dental pulp (17, 18). Data concerning the quantification of T lymphocytes in human dental pulp are inconsistent in the literature. Our results are in agreement with those of previous immunohistologic studies (7, 8, 13) but in contrast with those from flow cytometry analysis in which CD4+ T lymphocytes were 1.2 times more numerous than CD8+ T lymphocytes (12, 19). This discrepancy could be caused by differences in the selection method of T lymphocytes. In our experiment, we first eliminated dead cells, debris, and doublets. Then we combined a set of markers to obtain the percentage of cell subpopulations among leukocytes. Moreover, we used PBMCs as positive controls of the experiment. 4
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The CD4+/CD8+ ratio reflects the net regulatory balance of the CD3 population. According to Hahn et al (13), this ratio could be useful to know precisely the pulpal inflammatory status. Irreversible pulpitis was defined from a ratio of 1.14. In our study, the CD4+/CD8+ ratio was 0.5 in the healthy state of the pulp. Unlike T lymphocytes, B lymphocytes are not well recognized as normal residents of the dental pulp. They are not encountered or are rarely encountered in healthy human pulp (12, 13, 18). Our data showed a small but consistent percentage (1.65% 0.89%) of these cells. This discrepancy could be because of the detection technique. Immunologic studies have shown that interaction between T and B lymphocytes can be responsible for either the destruction or the protection of the pulp (6–8, 10). In our study, ratio B/CD3+ was around 0.05. This result is in agreement with Hahn et al (13), who found that more than 90% of lymphocytes were T lymphocytes in healthy pulp tissue. It is difficult to assign a significant role to B lymphocytes in healthy dental pulp. In addition to their main function of producing antibodies, B cells can also function as antigen-presenting cells, modulate DC functions, and produce immunoregulatory cytokines (20). Our data also showed that CD16+CD14 cells represented the most important population among the leukocytes (50.01% 9.08%, n = 7). These cells should be granulocytes, whereas CD45+CD14+ is compatible with the phenotype of monocytes and macrophages. A better characterization is needed to precisely characterize the granulocyte subtypes (neutrophils, eosinophils, or basophils) and macrophages. +
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Figure 3. Characterization of T helper, cytotoxic, and regulatory lymphocytes in healthy human dental pulp. (A) Flow cytometry analysis showing the phenotype of helper (CD45+CD3+CD4+) and cytotoxic (CD45+CD3+CD8+) T lymphocytes versus isotype controls. PBMCs were used as positive controls. Representative profiles of 1 experiment are shown. (B) Relative repartition of CD3+CD4+ versus CD3+CD8+ in CD45+ population in 5 different donors. (C) Flow cytometry analysis showing the phenotype of regulatory T cells (CD45+CD3+CD4+CD127low) expressing Foxp3 and CD25. A representative profile of 1 experiment is shown (n = 4).
NK cells play important roles in homeostasis, surveillance, and defense, the 3 major functions of the immune system (21). Quantitative analyses of NK cells are missing in human dental pulp except for deciduous teeth in which they were found by FACS to represent about 3% of the total cells (11, 22). Our data revealed that NK cells (CD3 CD56+) represent 2.63% 1.15% among leukocytes. This small percentage suggests that NK cells could be involved in pulp immunosurveillance, as described in other tissues. The difference with the study from Durut€urk et al (11) can be explained by the fact that deciduous and permanent teeth are different in their immunologic status because of pulp transformation into a granulation tissue that accompanies root resorption in deciduous teeth (23). In the present study, for the first time in healthy human dental pulp tissue, we found cells phenotypically compatible with regulatory T lymJOE — Volume -, Number -, - 2015
phocytes (CD45+CD3+CD4+CD127low) that express Foxp3 and CD25. Regulatory T cells interacting with antigen-presenting cells like DCs play an important role in regulating immune and inflammatory responses (24). In an immunohistologic study, the presence of Foxp3+ cells in human teeth with pulpitis was shown, whereas regulatory T cells were not detected in the healthy state (25). This result clearly indicates that healthy dental pulp is equipped for limiting or fine-tuning innate and adaptive responses even in the absence of pathogens. DCs are professional antigen-presenting cells that are critical for the induction of adaptive immunity and tolerance. In healthy dental pulp, they have been described to be present mostly in the paraodontoblastic region where they ensure immunosurveillance and capture of incoming antigens (2). In the present study, we found that DCs represent 4.51% 1.12% of the leukocyte population. DCs constitute a heterogeneous population in
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Figure 4. Identification of HLA-DRhigh HO-1+cells. (A) Flow cytometry analysis showing HLA-DR+ and HO-1+ cells in human dental pulp. A representative profile of 1 donor is shown (n = 4). (B) Confocal micrographs of cryostat sections from human dental pulp showing DAPI nucleic acid stain (blue fluorescence), HLADR+ (green fluorescence), HO-1+ cells (red fluorescence), and merged images with dual labeling (original magnification 63). Results are representative of 4 different dental pulps analyzed in the same manner.
various organs in the body that has been shown to display differences in terms of surface marker expression, functions, and origins (26). In the present study, we identified a specific subset of DCs expressing HO-1 (CD45+HLA-DRhigh HO-1+). HO-1 is a ubiquitously expressed, stressresponsive enzyme that catabolizes iron (Fe) protoporphyrin IX (ie, heme) into labile Fe, biliverdin, and carbon monoxide (CO). This enzyme protects cells against environmental stress including inflammatory and oxidative stress and inhibits the pathogenesis of several immunemediated inflammatory diseases. This action is caused by the immunoregulatory actions of HO-1, exerted on innate and adaptive immune cells (27, 28). In vitro studies with human dental pulp cell cultures have shown that HO-1 has a cytoprotective effect on these cells and contributes to odontoblast differentiation (29). However, HO-1 expression in professional antigen-presenting DCs had not been yet investigated regarding dental pulp. We can hypothesize that DCs expressing HO-1 should have immunoregulatory properties. In summary, our analysis of the phenotype of pulp leukocytes reveals interesting insights into the amount of immunocompetent cells and gives the immunologic mapping of leukocytes in healthy human dental pulp. Although this immune cell background obviously plays a role in immunosurveillance, it is reasonable to assume that the presence of regulatory T cells and DCs expressing HO-1 constitutes a specific cell environment suitable for immunoregulation. Better knowledge of the control of the immune response in dental pulp should lead to apparition of new therapeutic strategies in order to control the immune response, a preliminary step for vital pulp therapy.
Acknowledgments Alexis Gaudin and Emmanuelle Renard contributed equally to this work. The authors thank Dr Pascal Huet (maxillofacial surgeon) for providing extracted teeth. The authors deny any conflicts of interest related to this study. 6
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References 1. Cooper P, McLachlan J. Mediators of inflammation and regeneration. Adv Dent Res 2011;23:290–5. 2. Hahn C, Liewehr FR. Innate immune responses of the dental pulp to caries. J Endod 2007;33:643–51. 3. Hahn CL, Liewehr FR. Update on the adaptive immune responses of the dental pulp. J Endod 2007;33:773–81. 4. Staquet MJ, Carrouel F, Keller JF, et al. Pattern-recognition receptors in pulp defense. Adv Dent Res 2011;23:296–301. 5. Keller JF, Carrouel F, Colomb E, et al. Toll-like receptor 2 activation by lipoteichoic acid induces differential production of pro-inflammatory cytokines in human odontoblasts, dental pulp fibroblasts and immature dendritic cells. Immunobiology 2010;215:53–9. 6. Yoshiba N, Yoshiba K, Nakamura H, et al. Immunohistochemical localization of HLADR-positive cells in unerupted and erupted normal and carious human teeth. J Dent Res 1996;75:1585–9. 7. Jontell M, Gunraj MN, Bergenholtz G. Immunocompetent cells in the normal dental pulp. J Dent Res 1987;66:1149–53. 8. Izumi T, Kobayashi I, Okamura K, et al. Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth. Arch Oral Biol 1995;40:609–14. 9. Desai S, Love R, Rich A, et al. Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells? Int Endod J 2010;44:87–99. 10. Freitas P, Novaretti CP, Rodini CO, et al. Mast cells and lymphocyte subsets in pulps from healthy and carious human teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e95–102. 11. Durut€urk L, Sarı S, Seng€ul A. Immunocompetent cell level as a diagnostic reference for pulpal pathosis of primary teeth. Arch Oral Biol 2013;58:1517–22. 12. Mangkornkarn C, Steiner JC, Bohman R, et al. Flow cytometric analysis of human dental pulp tissue. J Endod 1991;17:49–53. 13. Hahn CL, Falkler WA Jr, Siegel MA. A study of T and B cells in pulpal pathosis. J Endod 1989;15:20–6. 14. Sakurai K, Okiji T, Suda H. Co-increase of nerve fibers and HLA-DR- and/or factor-XIIIa-expressing dendritic cells in dentinal caries-affected regions of the human dental pulp: an immunohistochemical study. J Dent Res 1999;78: 1596–608. 15. Saraswat M, Arya KV. Automated microscopic image analysis for leukocytes identification: a survey. Micron 2014;65:20–33. 16. Abbas A, Lichtman A. Effector Mechanisms of Cell-mediated Immunity. Cellular and Molecular Immunology, 5th ed. Philadelphia: Saunders; 2003:298–317.
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Clinical Research 17. Okiji T. Pulp as a connective tissue. In: Hargreaves KM, Goodis HE, Tay FR, eds. Seltzer and Bender’s Dental Pulp. Chicago: Quintessence; 2012:95–122. 18. Farges JC, Romeas A, Melin M, et al. TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer. J Dent Res 2003;82:652–6. 19. Angelova A, Takagi Y, Okiji T, et al. Immunocompetent cells in the pulp of human deciduous teeth. Arch Oral Biol 2004;49:29–36. 20. Bayry J, Lacroix-Desmazes S, Kazatchkine MD, et al. Modulation of dendritic cell maturation and function by B lymphocytes. J Immunol 2005;175:15–20. 21. Whiteside TL, Herberman RB. Role of Human natural killer cells in health and disease. Clin Diagn Lab Immunol 1994;1:125–33. 22. Sims¸ek S, Durut€urk L. A flow cytometric analysis of the biodefensive response of deciduous tooth pulp to carious stimuli during physiological root resorption. Arch Oral Biol 2005;50:461–8. 23. Harokopakis-Hajishengallis E. Physiologic root resorption in primary teeth: molecular and histological events. J Oral Sci 2007;49:1–12.
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24. Mahnke K, Bedke T, Enk AH. Regulatory conversation between antigen presenting cells and regulatory T cells enhance immune suppression. Cell Immunol 2007; 250:1–13. 25. Bruno K, Silva J, Silva T, et al. Characterization of inflammatory cell infiltrate in human dental pulpitis. Int Endod J 2010;43:1013–21. 26. Kushwah R, Hu J. Complexity of dendritic cell subsets and their function in the host immune system. Immunology 2011;133:409–19. 27. Soares MP, Marguti Ivo, Cunhaand Andreia, Larsen Rasmus. Immunoregulatory effects of HO-1: how does it work? Curr Opin Pharmacol 2009;9:482–9. 28. Mackern-Oberti JP, Riquelme S a, Llanos C, et al. Heme oxygenase-1 as a target for the design of gene and pharmaceutical therapies for autoimmune diseases. Curr Gene Ther 2014;14:218–35. 29. Lee YH, Lee NH, Bhattarai G, et al. Anti-inflammatory effect of pachymic acid promotes odontoblastic differentiation via HO-1 in dental pulp cells. Oral Dis 2013; 19:193–9.
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Phenotypic Analysis of Immunocompetent Cells in Healthy Human Dental Pulp Alexis Gaudin, DDS,*† Emmanuelle Renard, DDS,† Marcello Hill, MD, PhD,† Laurence Bouchet-Delbos, MEng,† Geraldine Bienvenu-Louvet, PhD,* Jean-Christophe Farges, DDS, PhD,‡ Maria-Cristina Cuturi, MD, PhD,† and Brigitte Alliot-Licht, DDS, PhD*† Abstract Introduction: Like other tissues in the body, the human dental pulp is equipped with a network of immune cells that can be mobilized against pathogens when they invade the tooth. Very little data, mostly obtained with classic histologic methods, have reported their quantities and relative percentages. The objective of this study was to characterize and precisely quantify immunocompetent cells in healthy human dental pulp by using fluorescence-activated cell sorting, together with identifying specific cell subsets in the leukocyte (CD45+) cells. Methods: Healthy human third molars were collected from 42 young patients. Dental pulps were separated from the hard tissues and prepared for flow cytometry or immunostaining analyses. Results: CD45+ cells represented 0.94% 0.65% of cells obtained from the enzymatic digestion of whole dental pulps (n = 34). CD16+CD14+ granulocytes/neutrophils (50.01% 9.08%, n = 7) were found to represent the major subpopulation in CD45+ cells followed by CD3+ T lymphocytes (32.58% 11%, n = 17), CD14+ monocytes (8.93% 5.8%, n = 7), and HLADRhigh Lin1- dendritic cells (4.51% 1.12%, n = 7). Minor subpopulations included CD3 CD56+ natural killer cells (2.63% 1.15%, n = 7) and CD19+ B lymphocytes (1.65% 0.89%, n = 17). We further identified cells harboring a phenotype compatible with Foxp3/CD25-expressing regulatory T lymphocytes (CD45+CD3+CD4+CD127low). Fluorescence-activated cell sorting analysis and confocal microscopy also revealed expression of HO-1 in HLA-DR+ cells. Conclusions: For the first time, this study identifies and precisely quantifies the relative proportion of immunocompetent cells potentially involved in tissue homeostasis of healthy human dental pulp. (J Endod 2015;-:1–7)
Key Words Fluorescence-activated cell sorting, healthy dental pulp, immune cells, immunosurveillance
H
uman dental pulp is a highly dynamic tissue equipped with a network of resident immunocompetent cells that are believed to play a major role in the maintenance of tissue homeostasis. Injuries such as dental caries, trauma, operative procedures, and periodontal diseases are susceptible to break this balance. Dental caries result from a complex process in which the composition of the oral bacterial biofilm evolves as the penetration of microorganisms through the enamel, dentin, and pulp occurs (1). Bacteria and their toxins present in dentinal tubules trigger several important cellular and molecular changes within the pulpal tissues. Like any other injured tissue in the body, dental pulp is able to mount innate adaptive immune responses intended to fight infection (2, 3). During bacterial penetration through the dentin, inflammatory mediators can be released by acidic degradation of the carious dentin and produced by resident pulp cells such as odontoblasts, pulpal fibroblasts, stem cells, endothelial cells, and/or tissue-resident immune cells (1, 4). Because of their peripheral localization, odontoblasts are considered the first line of cell defense of the pulp tissue. They express a variety of pathogen recognition receptors and chemokines and have been proposed to trigger the initial inflammatory and immune pulp response in cooperation with antigen-presenting dendritic cells (DCs) (5, 6). DCs, macrophages, T lymphocytes, natural killer (NK) cells, and B lymphocytes have been identified in inflamed human dental pulp based on histologic analysis (7, 8). Immune cells like DCs and T lymphocytes are also known to be resident in healthy pulp where they play a role in immunosurveillance. Immunosurveillance is a term used to describe the processes by which cells of the immune system look for and recognize foreign pathogens, such as bacteria and viruses, in the body. Immunosurveillance is well documented for skin, mucosa, lung, blood, and brain tissues. To our knowledge, precise characterization and quantification of immunocompetent cells in healthy human pulp have not been reported (2, 3). Fluorescence-activated cell sorting (FACS) is widely used to identify and characterize specific cell subsets in cellular suspensions obtained from tissues and is a great help in the determination of cell percentages in the tissues. Thus, FACS gives the opportunity to identify small cell populations with a set of different markers within the whole dental pulp tissue. Precise quantification, relative repartition among the leucocyte population, and detection of specific immunoregulatory cells will lead to a better understanding of the pulp initial capacity to mount efficient immune responses and regulate them. This knowledge is important to better
From the *Faculty of Odontology, University of Nantes, Nantes, France; †INSERM, Institut de Transplantation et de Recherche en Transplantation, Nantes, France; and ‡Laboratory of Tissue Biology and Therapeutic Engineering, University of Lyon, Lyon, France. Address requests for reprints to Dr Alexis Gaudin, Faculte de Chirurgie Dentaire, 1 Place Alexis Ricordeau, 44042 Nantes, Cedex 1, France. E-mail address: alexis. [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.01.005
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Phenotypic Analysis of Immunocompetent Cells
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Clinical Research understand the physiopathology of the pulp during bacterial aggression. Using FACS analysis, our study aimed to characterize and precisely quantify immunocompetent cells and identify specific subsets among the leukocytes (CD45+ cells) present in healthy human dental pulp. Furthermore, the detection of subsets involved in immunoregulation should lead to the discovery of targets for future therapeutic strategies.
Materials and Methods Collection of Pulp Samples Tissue samples were obtained from 42 healthy patients between the ages of 11 and 25 years who were elected to have extractions of healthy third molars under general anesthesia for orthodontic reasons. Teeth were collected with patients’ (or the parents of patients) informed consent. The surgeries were performed at the Clinic of Stomatology of Nantes under approved guidelines. Impacted or embedded teeth that were cut during removal were excluded. Freshly extracted molars were taken directly from the surgical site and placed into sterile phosphate buffered saline (PBS) (pH = 7.4). Attached soft tissue was removed from the root surface by sterile scalpel blades. They were then cracked open, and the pulp tissue was gently removed with cotton pliers and placed in a Petri dish containing 1 mL PBS. Tissue Preparation Pulp tissues were minced into small pieces (0.5 mm3) using sterile scalpel blades, transferred into a 50-mL sterile tube, and centrifuged at 1500 rpm for 10 minutes. The supernatant was removed, and the tissue pellet was digested in 5 mL collagenase D (Roche, Meylan, France) and then incubated at 37 for 45 minutes. The pulp tissue was completely dissociated in 30 to 45 minutes. EDTA (10 mmol/L) was added for the last 5 minutes to stop the enzymatic digestion. The cell suspension was washed twice with 50 mL sterile PBS, passed through a 100-mm nylon mesh filter, and resuspended in 2 mL PBS supplemented with 2 mmol/L EDTA and 2% fetal calf serum (FCS). We routinely obtained 1 to 7 million cells per patient, which were subsequently used for antibody staining. Flow Cytometry For cell surface staining, suspensions of single pulp cells were distributed into 96-well V-bottomed plates, washed, and incubated in PBS/2% FCS/0.2% azide. According to the manufacturer’s recommendations, cells were incubated on ice for 30 minutes with anti-CD45 PECy7 (clone HI-30, mouse immunoglobulin [Ig] G1) and monoclonal conjugated antibodies (mAbs [Table 1]) or isotype controls. The following combinations were used to identify pulp immunocompetent cells: B lymphocytes: CD45/CD19; T lymphocytes: CD45/CD3/CD4/
CD8; NK cells: CD45/CD3/CD56; DCs: CD45/HLA-DR/Lin1; and monocytes, granulocytes, and macrophages: CD45/CD14/CD16. Single-cell suspensions were washed twice with PBS/0.2% FCS/0.1% azide before fixation with 2% paraformaldehyde and then maintained in the dark at 2 C–8 C until further use. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of other patients by Ficoll-Hypaque density gradient centrifugation (Amersham, Les Ulis, France) in order to be used as positive controls. Flow cytometry analysis was performed by using the BD FACS Canto Flow Cytometer (Becton Dickinson, San Jose, CA). Results were analyzed using FlowJo analysis software (Tree Star, Ashland, OR). Primary gates based on physical parameters (forward and side light scatter) and fixable viability dye eFluor450 (eBioscience, Paris, France) were used in all experiments to exclude dead cells or debris. For intracellular staining, cells were distributed into 96-well Vbottomed plates, washed, and incubated in PBS 0.5% saponin for 20 minutes on ice. After 3 washes in PBS 0.1% saponin, cells were incubated for 30 minutes on ice according to the manufacturer’s recommendations with anti-CD45 PE-Cy7 (clone HI-30, mouse IgG1) and mAbs (Table 1) or isotype controls. The following mAb combinations were used: DCs–heme oxygenase-1 (HO-1): CD45/HLA-DR, regulatory T lymphocytes (Tregs) CD45/CD3/CD4/CD127/Foxp3, and CD45/CD3/ CD127/CD4/CD25. After 2 washes with PBS/0.2% FCS/0.1% saponin and 1 wash with PBS/2% FCS/0.2% azide, cells were fixed with 2% paraformaldehyde and analyzed as previously described.
Immunohistology and Confocal Image Analysis For immunohistology, third molar dental pulps from healthy donors were frozen and cryosectioned (10 mm). Tissue sections were fixed with acetone and saturated using PBS/4% bovine serum albumin/10% goat serum. Slides were then permeabilized with 0.5% saponin and stained with anti–HO-1 primary mAb (Stressgen, San Diego, CA). Slides were then incubated with biotinylated conjugated mAb diluted at 1/500. A second incubation was performed with streptavidin 568 conjugated biot mAb diluted 1/1000. Cells were also stained with anti-HLA-DR FITC mAb (BD Biosciences, Le Pont de Claix, France). Matching isotype control mouse antibodies were included. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Slides were mounted in ProLong AntiFade reagent (Molecular Probes, Saint-Aubin, France) and analyzed with a Leica confocal microscope (Leica Microsystems, Heidelberg, Germany) and Leica TCS NT software.
Results For the first time, we propose a precise quantification of leukocytes within the healthy human dental pup. Cellular suspensions were obtained
TABLE 1. Monoclonal Antibodies Used in This Study Antibodies CD45 CD19 CD3 CD4 CD8 HLA-DR Lin1 CD14 CD16 HO-1 CD127 CD25 Foxp3
Fluorochrome
Isotype
Clone
Manufacturer
PE-Cy7 PE APC-Cy7 PerCP-Cy5.5 FITC V500 FITC PE FITC FITC PE FITC APC
Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG2a Mouse IgG2b Mouse IgG2a Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1 Rat Ig2a
HI-30 HIB19 SK7 L200 HIT8a G46-6 NCAM16.2 M5E2 3G8 HO-1-1 HIL-7R-m21 M-A251 PCH101
BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences Abcam, Cambridge, UK BD Biosciences BD Biosciences eBioscience
Ig, immunoglobulin.
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Figure 1. Quantification of leukocytes (CD45+) in healthy human dental pulp. (A) The gating strategy used for the elimination of dead cells, debris, and doublets. Representative profiles of 1 experiment are shown. (B) Flow cytometry analysis showing cells stained with CD45 antibody or the appropriate fluorochromeconjugated control IgG. (C) The percentage of CD45+ represented 0.94% 0.65% of cells obtained after enzymatic digestion of the whole healthy human dental pulp (n = 34).
after enzymatic digestion, and all the results in this study were based on the same gating strategy to generate comparable data (Fig. 1A). Debris, nonviable cells, and doublets were excluded. Viable cells were found to represent 84.35% 5.8% of the whole pulp cells (n = 34) (data not shown). The percentage of leukocytes (CD45-expressing cells) was 0.94% 0.65% of the total viable pulp cells (Fig. 1B). Results were similar for the 34 different donors tested (Fig. 1C). We then investigated the expression of the phenotypic markers CD19, CD3, CD56, HLA-DR CD14, and CD16 by CD45+ leukocytes to determine the relative proportions of the major subsets of immunocompetent cells (Fig. 2A and B). We found that CD16+CD14+ granulocytes/ neutrophils represent the major subpopulation among CD45 + cells (50.01% 9.08%, n = 7) followed by CD3+ T cells (32.58% 11%, n = 17), CD14+ monocytes (8.93% 5.8%, n = 7), and HLA-DRhigh Lin1- DCs (4.51% 1.12%, n = 7). Minor populations included CD3 CD56+ NK cells (2.63% 1.15%, n = 7) and CD19+ B lymphocytes (1.65% 0.89%, n = 17). This repartition was homogenous among the donors tested. Lymphocytes (CD45+CD3+) are a major component of key immunocompetent cells. The lymphocyte population is mainly composed of CD4+ helper (or regulatory) and CD8+ cytotoxic TCRab+ T cells. The CD45+CD3+ lymphocyte population can also include NK T cells, mucosa-associated invariant T cells, or TCRgd+ T cells. Helper and cytotoxic lymphocytes were clearly identified by our flow cytometry analysis (Fig. 3A). Our data suggested that the helper T lymphocyte population (CD45+CD3+CD4+) was smaller than the cytotoxic T lymphocyte population (CD45+CD3+CD8+) (11.18% 0.5% vs 21.6% 2.8%, respectively), resulting in a CD4/CD8 ratio of 0.51. JOE — Volume -, Number -, - 2015
This ratio is different than the ratio obtained on PBMCs (Fig. 3B). Furthermore, we identified cells harboring a phenotype compatible with regulatory T lymphocytes (CD45+CD3+CD4+CD127low) expressing Foxp3 and CD25 (Fig. 3C). We also quantified the proportion of NK T lymphocytes (CD45+CD3+CD56+) and found that these cells represented 2.45% 1.34% of the total CD45+ cells (data not shown). We finally examined whether DCs possess immunoregulatory components in healthy dental pulp. We found HLA-DR+ cells expressing the enzyme HO-1 (Fig. 4A). Confocal microscopy showed the cytoplasmic expression of HO-1 in the HLA-DR+ cells (Fig. 4B).
Discussion Peripheral organs, such as the skin, gastrointestinal tract, and lungs, are barriers against invading microorganisms. In the healthy state, these organs contain sentinels cells including DCs, macrophages, and T lymphocytes to counteract pathogen invasion upon barrier breach within these organs. Dental pulp also contains a variety of immunocompetent cells (9–11). However, a better understanding of this background is necessary to understand the steady state of the pulp before comparison with inflamed tissue. In the field of endodontic research, Mangkornkarn et al (12) were the first to use FACS to examine the cellular components of human dental pulp tissue. FACS is a highly sensitive tool for cell counting compared with immunolabeling of histologic sections (7, 8, 12–14). FACS analysis gives the opportunity to combine several antibodies and to use positive or negative selection methods to identify and quantify specific cell populations in a reliable way (15). In this study, we
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Figure 2. Relative repartition of key immunocompetent cells among the CD45 + population. (A) Flow cytometry histograms of specific markers (black histogram), CD19, CD3, CD56, HLA-DR, CD14, and CD16, expressed among the CD45+ cells versus isotype controls (gray histogram) of 1 representative patient. Numbers represent the percentage of positive cells. (B) The level of expression of the following phenotypic markers: CD19+ for B lymphocytes, CD3+ for T lymphocytes, CD3 CD56+ for NKs, HLA-DRhighlin1- for dendritic cells, CD14+ for monocytes and macrophages, and CD16+CD14 for others (granulocytes and neutrophils) among CD45+ cells. The pie chart summarizes the relative repartition of key immunocompetent cells among the CD45 + population.
wanted to characterize the distribution of the cell subtypes within the leukocyte population present in healthy human dental pulp. Leukocytes were found to represent less than 1% of the cell population obtained after enzymatic digestion of the whole pulp tissue. These cells function to detect pathogens, allergens, and other harmful environmental agents that penetrate into the body (16). In our experiments, teeth had never been in contact with pathogens because they were extracted before any contact with the oral cavity. The percentage of leukocytes is relatively high for healthy connective tissue (16). This indicates that dental pulp contains the proper armamentarium to detect future pathogens. In our study, we found that CD8+ T lymphocytes (21.6% 2.8%) outnumbered CD4+ T lymphocytes (11.18% 0.5%) in healthy conditions. T lymphocytes are recognized as normal resident cells of human and rat dental pulp (17, 18). Data concerning the quantification of T lymphocytes in human dental pulp are inconsistent in the literature. Our results are in agreement with those of previous immunohistologic studies (7, 8, 13) but in contrast with those from flow cytometry analysis in which CD4+ T lymphocytes were 1.2 times more numerous than CD8+ T lymphocytes (12, 19). This discrepancy could be caused by differences in the selection method of T lymphocytes. In our experiment, we first eliminated dead cells, debris, and doublets. Then we combined a set of markers to obtain the percentage of cell subpopulations among leukocytes. Moreover, we used PBMCs as positive controls of the experiment. 4
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The CD4+/CD8+ ratio reflects the net regulatory balance of the CD3 population. According to Hahn et al (13), this ratio could be useful to know precisely the pulpal inflammatory status. Irreversible pulpitis was defined from a ratio of 1.14. In our study, the CD4+/CD8+ ratio was 0.5 in the healthy state of the pulp. Unlike T lymphocytes, B lymphocytes are not well recognized as normal residents of the dental pulp. They are not encountered or are rarely encountered in healthy human pulp (12, 13, 18). Our data showed a small but consistent percentage (1.65% 0.89%) of these cells. This discrepancy could be because of the detection technique. Immunologic studies have shown that interaction between T and B lymphocytes can be responsible for either the destruction or the protection of the pulp (6–8, 10). In our study, ratio B/CD3+ was around 0.05. This result is in agreement with Hahn et al (13), who found that more than 90% of lymphocytes were T lymphocytes in healthy pulp tissue. It is difficult to assign a significant role to B lymphocytes in healthy dental pulp. In addition to their main function of producing antibodies, B cells can also function as antigen-presenting cells, modulate DC functions, and produce immunoregulatory cytokines (20). Our data also showed that CD16+CD14 cells represented the most important population among the leukocytes (50.01% 9.08%, n = 7). These cells should be granulocytes, whereas CD45+CD14+ is compatible with the phenotype of monocytes and macrophages. A better characterization is needed to precisely characterize the granulocyte subtypes (neutrophils, eosinophils, or basophils) and macrophages. +
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Figure 3. Characterization of T helper, cytotoxic, and regulatory lymphocytes in healthy human dental pulp. (A) Flow cytometry analysis showing the phenotype of helper (CD45+CD3+CD4+) and cytotoxic (CD45+CD3+CD8+) T lymphocytes versus isotype controls. PBMCs were used as positive controls. Representative profiles of 1 experiment are shown. (B) Relative repartition of CD3+CD4+ versus CD3+CD8+ in CD45+ population in 5 different donors. (C) Flow cytometry analysis showing the phenotype of regulatory T cells (CD45+CD3+CD4+CD127low) expressing Foxp3 and CD25. A representative profile of 1 experiment is shown (n = 4).
NK cells play important roles in homeostasis, surveillance, and defense, the 3 major functions of the immune system (21). Quantitative analyses of NK cells are missing in human dental pulp except for deciduous teeth in which they were found by FACS to represent about 3% of the total cells (11, 22). Our data revealed that NK cells (CD3 CD56+) represent 2.63% 1.15% among leukocytes. This small percentage suggests that NK cells could be involved in pulp immunosurveillance, as described in other tissues. The difference with the study from Durut€urk et al (11) can be explained by the fact that deciduous and permanent teeth are different in their immunologic status because of pulp transformation into a granulation tissue that accompanies root resorption in deciduous teeth (23). In the present study, for the first time in healthy human dental pulp tissue, we found cells phenotypically compatible with regulatory T lymJOE — Volume -, Number -, - 2015
phocytes (CD45+CD3+CD4+CD127low) that express Foxp3 and CD25. Regulatory T cells interacting with antigen-presenting cells like DCs play an important role in regulating immune and inflammatory responses (24). In an immunohistologic study, the presence of Foxp3+ cells in human teeth with pulpitis was shown, whereas regulatory T cells were not detected in the healthy state (25). This result clearly indicates that healthy dental pulp is equipped for limiting or fine-tuning innate and adaptive responses even in the absence of pathogens. DCs are professional antigen-presenting cells that are critical for the induction of adaptive immunity and tolerance. In healthy dental pulp, they have been described to be present mostly in the paraodontoblastic region where they ensure immunosurveillance and capture of incoming antigens (2). In the present study, we found that DCs represent 4.51% 1.12% of the leukocyte population. DCs constitute a heterogeneous population in
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Figure 4. Identification of HLA-DRhigh HO-1+cells. (A) Flow cytometry analysis showing HLA-DR+ and HO-1+ cells in human dental pulp. A representative profile of 1 donor is shown (n = 4). (B) Confocal micrographs of cryostat sections from human dental pulp showing DAPI nucleic acid stain (blue fluorescence), HLADR+ (green fluorescence), HO-1+ cells (red fluorescence), and merged images with dual labeling (original magnification 63). Results are representative of 4 different dental pulps analyzed in the same manner.
various organs in the body that has been shown to display differences in terms of surface marker expression, functions, and origins (26). In the present study, we identified a specific subset of DCs expressing HO-1 (CD45+HLA-DRhigh HO-1+). HO-1 is a ubiquitously expressed, stressresponsive enzyme that catabolizes iron (Fe) protoporphyrin IX (ie, heme) into labile Fe, biliverdin, and carbon monoxide (CO). This enzyme protects cells against environmental stress including inflammatory and oxidative stress and inhibits the pathogenesis of several immunemediated inflammatory diseases. This action is caused by the immunoregulatory actions of HO-1, exerted on innate and adaptive immune cells (27, 28). In vitro studies with human dental pulp cell cultures have shown that HO-1 has a cytoprotective effect on these cells and contributes to odontoblast differentiation (29). However, HO-1 expression in professional antigen-presenting DCs had not been yet investigated regarding dental pulp. We can hypothesize that DCs expressing HO-1 should have immunoregulatory properties. In summary, our analysis of the phenotype of pulp leukocytes reveals interesting insights into the amount of immunocompetent cells and gives the immunologic mapping of leukocytes in healthy human dental pulp. Although this immune cell background obviously plays a role in immunosurveillance, it is reasonable to assume that the presence of regulatory T cells and DCs expressing HO-1 constitutes a specific cell environment suitable for immunoregulation. Better knowledge of the control of the immune response in dental pulp should lead to apparition of new therapeutic strategies in order to control the immune response, a preliminary step for vital pulp therapy.
Acknowledgments Alexis Gaudin and Emmanuelle Renard contributed equally to this work. The authors thank Dr Pascal Huet (maxillofacial surgeon) for providing extracted teeth. The authors deny any conflicts of interest related to this study. 6
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