J Med Microbiol 54 (2005), 413-416; DOI: 10.1099/jmm.0.45728-0
© 2005 Society for General Microbiology
ISSN 0022-2615
Bacteria recovered from dental pulp induce apoptosis of lymph node cells
A P Ribeiro-Sobrinho1,
F LA Rabelo2,
C BO Figueiredo1,
J I Alvarez-Leite2,
J R Nicoli3,
M Uzeda4 and
L Q Vieira2
1,2,3Dental School1, Department of Biochemistry and Immunology/ICB2 and Department of Microbiology/ICB3, Universidade Federal de Minas Gerais (UFMG), CP 486, 30161-970, Belo Horizonte, Brazil 4Department of Microbiology – Institute of Microbiology ‘Professor Paulo de Góes', UFRJ, Rio de Janeiro, Brazil
Correspondence L. Q. Vieira lqvieira{at}icb.ufmg.br
Received April 30, 2004
Accepted December 15, 2004
Apoptosis is critical in the pathogenesis of several infectious diseases. The induction of apoptosis was assessed in mouse lymph node cells by four bacteria recovered from infected human dental pulp: Gemella morbillorum, Clostridium butyricum, Fusobacterium nucleatum and Bifidobacterium adolescentis. Smaller lymph nodes and smaller numbers of cells were observed after experimental dental pulp infection with C. butyricum, suggesting that this bacterium induces cell death. Apoptosis was evaluated by determination of cell ploidy and detection of DNA degradation in cells cultured with killed bacteria. Paraformaldehyde-killed C. butyricum and heat-killed G. morbillorum induced substantial cell death, while F. nucleatum and B. adolescentis induced cell death at lower levels. No bacterial preparations induced apoptosis in cells from mice genetically deficient for tumour necrosis factor receptor p55 (TNFRp55), implicating this receptor directly or indirectly as a mediator in the process. It was concluded that apoptosis may be induced during periapical lesions of pulpal origin.
Abbreviations: PI, propidium iodide; TNF, tumour necrosis factor.
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INTRODUCTION
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The interaction between infectious agents and the host defence system is the key element in determining health or disease. Accordingly, periapical bone destruction subsequent to dental pulp infections is thought to be caused by colonization by pathogenic bacteria and consequent triggering of host defence mechanisms (Kakehashi et al., 1965). Many questions concerning the mechanisms involved in the pathogenesis of periapical lesions have been answered. Still, there are many doubts that must be clarified, such as the capacity of dental pulp infection to promote apoptosis and the effects of apoptosis on the aetiopathogenesis of this disease. The strategies used by pathogens to activate programmed cell death may subvert normal host defence responses. Accordingly, activation of death of immune cells by the oral bacterium Fusobacterium nucleatum might represent one mechanism by which this bacterium mediates immunosuppression and inactivation of potentially responsive cells (Jewett et al., 2000). Here, we describe a reduction in the number of lymph node cells during experimental dental pulp infection in germ-free mice. The bacteria studied induced different levels of apoptotic cell death and the apoptosis-inducing molecules were heat- or paraformaldehyde-labile. Apoptosis of lymph node cells was dependent on the presence of the tumour necrosis factor (TNF) receptor p55 (TNFRp55).
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METHODS
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Animals.
Germ-free Swiss/NIH mice (Gnotobiology Laboratory, ICB, UFMG, Belo Horizonte, Brazil) were used to perform the in vivo experiments (Ribeiro Sobrinho et al., 2001, 2002). Lymph node cells recovered from conventional Swiss/NIH mice (Gnotobiology Laboratory, ICB, UFMG) were used to perform the in vivo experiments (Ribeiro Sobrinho et al., 2001, 2002), while C57BL/6 mice (CEBIO, UFMG) and gene knockout mice for the p55 receptor for TNF (p55–/–; a kind gift from Dr Klaus Pfeffer, Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Germany) on a C57BL/6 background (Pfeffer et al., 1993) were used for the in vitro apoptosis assessments. Experimental protocols and care of animals were in accordance with the institutional requirements for humane treatment.
Bacteria.
The bacteria used in this study were recovered from two patients with pulpal necrosis treated at the Endodontic Clinic of the Dental School, UFMG (Lanna et al., 2001; Ribeiro Sobrinho et al., 2001). The bacteria were Gemella morbillorum from patient 1 and Bifidobacterium adolescentis, F. nucleatum and Clostridium butyricum from patient 2. Samples were stored at –70 °C and recovered, when necessary, in brain heart infusion (BHI) broth (Difco) supplemented and pre-reduced (BHI-S-PRAS), plated on to Petri dishes containing blood agar supplemented with haemin and menadione (Sutter et al., 1980) and incubated at 37 °C inside an anaerobic chamber (Forma Scientific Co.).
Bacterial inoculation and its effect on murine lymphocytes.
The bacterial inoculum was dripped into the germ-free mouse root canal using methodology previously described (Ribeiro Sobrinho et al., 2001). Before any surgical procedure, animals were anaesthetized intraperitoneally using pentobarbital (35 mg kg–1) with fentanil (25 µg kg–1) (Cristalia). The inferior right incisor was selected for our studies. The tooth was trepanned with a steel file (33
; Maillefer) in low rotation. The remaining pulp was removed using an exploring probe and the root canal system was dried with sterile absorbent paper tips. The inoculum was dripped into the instrumented root canal, with 1 ml syringes and 26G (13 x 4.5) needles (Injex Indústrias Cirúrgicas LTDA). Bacteria were inoculated separately or together (i.e. B. adolescentis, F. nucleatum and C. butyricum as found in patient 2). After inoculation, the orifice of the root canal was sealed with paraffin. Non-infected controls were also analysed. Mice were sacrificed on days 10 and 20 after inoculation. The submandibular lymph nodes adjacent to the tooth submitted to surgical procedures were removed and single-cell suspensions in RPMI 1640 (Sigma) were obtained. Since the number of cells recovered from the submandibular lymph nodes from individual animals was not enough to obtain a satisfactory number of culture wells to be stimulated, organs from different animals subjected to the same stimulus (n = 5) were pooled. Each experiment was repeated four times and the results pooled.
Bacterial preparations.
Bacterial samples grown in BHI-S-PRAS for 48 h at 37 °C were adjusted to a concentration of 1.0 x 109– 5 x 109 c.f.u. ml–1, washed once with 4 ml sterile PBS (pH 7.0) and the pellet was resuspended in 4 ml PBS. The bacteria were either treated with 1 % paraformaldehyde (Jewett et al., 2000) or heat-killed (Ribeiro Sobrinho et al., 2002). Viability of cells was tested on BHI agar. Live bacteria were not used in vitro because they would grow in the cell culture and might deplete the medium of nutrients, thus affecting the response of mouse cells.
Cell cultures.
Cells were cultured with bacterial stimuli as described previously (Ribeiro Sobrinho et al., 2002).
Determination of DNA content.
Cells were stained with propidium iodide (PI) using a method described previously (Nicoletti et al., 1991) with modifications. Cells were washed twice with cold PBS and resuspended in 750 µl of a hypotonic solution containing PI (0.1 % sodium citrate, 0.005 % Triton X-100, 50 µg PI ml–1). After 12–18 h of incubation at 4 °C in the dark, cells were analysed using a flow cytometer (FACSVantage; Becton Dickinson). Cells undergoing apoptosis showed a lower PI fluorescence intensity (DNA content) when compared with viable cells due to DNA fragmentation, typical of apoptosis. These cells were termed subdiploid. The apoptosis ratio was calculated as % subdiploid cells in treated group/% subdiploid cells in control group.
Detection of DNA fragmentation by gel electrophoresis.
DNA isolation from 1 x 107 pooled lymph node cells cultured in the presence or absence of bacterial stimuli was performed as described previously (Kurita-Ochiai et al., 1999). Samples were subjected to horizontal gel electrophoresis in a 1.5 % agarose gel containing 0.71 µg ethidium bromide ml–1.
Statistical analysis of data.
Statistical analysis was carried out using Student's non-paired t-test. The null hypothesis was rejected when P < 0.05.
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RESULTS AND DISCUSSION
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Apoptosis is involved in many physiological processes including the immune response (Schwartzman & Cidlowski, 1993; King & Cidlowski, 1998). Inducers of apoptosis include TNF and bacterial products (Fraser & Evan, 1996; Nagata, 1997; Thompson, 1995; Weinrauch & Zychlinsky, 1999). Periapical diseases vary widely in severity among individuals, and it is reasonable to propose that this variability is due to differences in the composition of the microbiota as well as in the immune responses against the oral microbiota.
Bacteria (G. morbillorum from patient 1 and B. adolescentis, F. nucleatum and C. butyricum from patient 2) were inoculated into the root canals of germ-free mice. At 10 and 20 days after inoculation, the submandibular lymph nodes adjacent to the teeth submitted to surgical procedures were aseptically removed and the cells counted. At both time points, mice infected with C. butyricum alone or together with F. nucleatum and B. adolescentis presented smaller lymph nodes than the control group and the other mice infected with a single bacterial species (data not shown). Accordingly, smaller numbers of cells were recovered from lymph nodes from mice infected with C. butyricum (Fig. 1). One possible reason for the smaller lymph nodes after infection with C. butyricum was the triggering of cell death by the bacteria. Hence, we investigated whether apoptosis of lymph node cells would take place following exposure to the isolated bacteria. Lymph node cells from Swiss/NIH mice were exposed in vitro to paraformaldehyde-treated or heat-killed bacterial preparations. Addition of heat-killed G. morbillorum or paraformaldehyde-treated C. butyricum (the latter both when added alone and in combination with F. nucleatum and B. adolescentis) to lymph node cells from Swiss/NIH mice in culture induced high levels of apoptotic cell death, as determined by flow cytometry analysis of PI-stained cells (Fig. 2a). Paraformaldehyde-treated and heat-killed F. nucleatum and B. adolescentis induced similar levels of apoptotic cell death to the control cultures, as shown by the ratio of approximately 1 (Fig. 2a). In cultures where there was an increase in apoptotic cell death, we also found a parallel decrease in non-apoptotic cells (Fig. 3a). Interestingly, apoptosis induced by heat-killed C. butyricum or paraformaldehyde-killed G. morbillorum was marginal. Confirmation of apoptosis was obtained by DNA gel electrophoresis (Fig. 4) and Giemsa staining (data not shown). Heat-killed G. morbillorum and paraformaldehyde-killed F. nucleatum and C. butyricum induced DNA fragmentation of lymphocyte cells (Fig. 4). C. butyricum and G. morbillorum translocate to the draining lymph nodes at higher rates than other dental pulp bacteria studied (Ribeiro Sobrinho et al., 2001). It is possible that the apoptotic cell death observed here facilitates translocation to the draining lymph nodes by impairing host defences.
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Fig. 1. Number of cells recovered from submandibular lymph nodes from Swiss/NIH germ-free mice in the absence (open bars) or presence (filled bars) of infection on days 10 (a) and 20 (b) after inoculation. Experiments were performed independently. Lymph node cells from five mice were pooled. Each experiment (five mice per group per experiment) was repeated four times. Bars represent the means of individual experiments ± SD. *, Statistical differences between the mock-infected and infected groups (P < 0.05). Association, infection with B. adolescentis, F. nucleatum and C. butyricum.
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Fig. 2. Apoptosis induced by incubation of lymph node cells with dental pulp bacteria. Lymph node cells from Swiss/NIH (a) or p55 gene knockout (b) mice were incubated with equal numbers of paraformaldehyde-treated (open bars) or heat-killed (filled bars) bacteria at a 10 : 1 bacterium : cell ratio for 24 h. Bars represent the pool of lymph nodes from five mice. Results are from one representative experiment of three performed. The apoptosis ratio was calculated as % subdiploid cells in treated group/% subdiploid cells in the control group.
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Fig. 3. DNA analysis performed by flow cytometry of lymph node cells from Swiss/NIH (a) and TNFRp55–/– (b) mice. Untreated (control) or stimulated cells (H, heat-killed; P, paraformaldehyde-treated) were analysed as described in Methods. DNA content considered consistent with viable and apoptotic cells is indicated by horizontal lines. Results are from one representative experiment of three.
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Fig. 4. Gel electrophoresis of DNA from cells from C57BL/6 mice co-cultured with a bacteria : cell ratio of 10 : 1. DNA extracted from cells was loaded on to a 2 % agarose gel and submitted to electrophoresis. Lanes: 1, molecular size marker; 2, untreated cells; 3, cells co-cultured with paraformaldehyde-killed F. nucleatum; 4, cells co-cultured with paraformaldehyde-killed C. butyricum; 5, cells co-cultured with paraformaldehyde-killed B. adolescentis; 6, cells co-cultured with heat-killed G. morbillorum. The results of one experiment of four performed are shown, two with lymph node cells from Swiss/NIH mice and two with lymph node cells from C57BL/6 mice, with identical results.
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Numerous mechanisms are used by extracellular and intracellular pathogenic bacteria to induce apoptosis in host cells (Mahida et al., 1996; Morimoto & Bonavida, 1992) including pore-forming toxins, other toxins that express their enzymic activity in the host cytosol, effector proteins delivered directly into host cells by a highly specialized type III secretory system, superantigens and other modulators of host-cell death (Ikigai & Nakae, 1987; Stevens, 1997; Weinrauch & Zychlinsky, 1999). The best-characterized TNF receptor-like death receptors are CD95 (also called Fas or Apo1) and TNFRp55 (also called TNFR1 or CD120a) (Smith et al., 1994; Nagata, 1997). Thus, we investigated the role of TNFRp55 in the induction of apoptosis under our experimental conditions. Incubation of lymph node cells recovered from p55 gene knockout mice with heat-killed G. morbillorum and paraformaldehyde-killed C. butyricum induced significantly lower levels of cell death than those observed in Swiss/NIH cells (Figs 2 and 3). Thus, the dental pulp bacterial preparations used in this work triggered the process of cell death and involved TNFRp55. The mechanism by which G. morbillorum and C. butyricum induced apoptosis in our model is not completely understood: under our conditions, apoptosis was dependent on the presence of TNFRp55; however, whether our bacterial preparations were inducing TNF, which induces apoptosis, or whether a molecule derived from the bacteria was directly binding to the receptor, was not known. In addition, the role of TNFRp55 may be indirect. The bacterial apoptosis-inducing molecules present were paraformaldehyde-sensitive in G. morbillorum and heat-labile in C. butyricum. It is highly likely that TNF is being induced in these cultures, since bacteria are potent inducers of TNF production.
Jewett et al. (2000) found that only paraformaldehyde-treated F. nucleatum was capable of inducing the death of human peripheral blood mononuclear cells, while this ability was lost when the bacterium was killed by heat treatment. The levels of apoptosis described by these authors were similar to the levels seen here, and were far below the levels of apoptosis induced by G. morbillorum and C. butyricum. Based on our previous (Ribeiro Sobrinho et al., 2001, 2002) and recent findings, we suggest that dental pulp bacteria that induce apoptotic death of lymphocytes affect the outcome of periradicular disease by inducing killing of immunocompetent cells, either effectors of resistance to infection or modulators of the immune response. It is possible that apoptosis happens during human dental pulp infection, given the in vitro and in vivo evidence presented herein.
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ACKNOWLEDGEMENTS
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This study was supported by CNPq, FAPEMIG and PRPq-UFMG. The technical help of Maria Gorete Barbosa Ribas, Ronilda Maria de Paula (in memoriam) and Maria Helena de Oliveira are acknowledged. We wish to thank Dr José Miguel Ortega for useful discussions.
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REFERENCES
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|---|
Fraser, A. & Evan, G. A. (1996). A license to kill. Cell 85, 781–784.[CrossRef][Medline]
Ikigai, H. & Nakae, T. (1987). Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane. J Biol Chem 262, 2150–2155.[Abstract/Free Full Text]
Jewett, A., Hume, W. R., Le, H., Huynh, T. N., Han, Y. W., Cheng, G. & Shi, W. (2000). Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum. Infect Immun 68, 1893–1898.[Abstract/Free Full Text]
Kakehashi, S., Stanley, R. H. & Fitzgerald, R. J. (1965). The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 20, 340–349.[CrossRef][Medline]
King, K. L. & Cidlowski, J. A. (1998). Cell cycle regulation and apoptosis. Annu Rev Physiol 60, 601–617.[CrossRef][Medline]
Kurita-Ochiai, T., Fukushima, K. & Ochiai, K. (1999). Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells. Infect Immun 67, 22–29.[Abstract/Free Full Text]
Lanna, M. A., Ribeiro-Sobrinho, A. P., Stehling, R., Garcia, G. D., Silva, B. K. C., Hamdan, J. S., Nicoli, J. R., Carvalho, M. A. R. & Farias, L. M. (2001). Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro. Oral Microbiol Immunol 16, 100–105.[CrossRef][Medline]
Mahida, Y. R., Makh, S., Hyde, S., Gray, T. & Borriello, S. P. (1996). Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment. Gut 38, 337–347.[Abstract/Free Full Text]
Morimoto, H. & Bonavida, S. (1992). Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis.ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha. J Immunol 149, 2089–2094.[Abstract]
Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355–365.[CrossRef][Medline]
Nicoletti, J., Migliorati, G., Pagliacci, M. C., Grignani, F. & Riccardi, C. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139, 271–279.[CrossRef][Medline]
Pfeffer, K., Matsuyama, T., Kundig, T. M. & 7 other authors (1993). Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L.monocytogenes infection. Cell 73, 457–467.[CrossRef][Medline]
Ribeiro Sobrinho, A. P., Lanna, M. A., Farias, L. M., Carvalho, M. A. R., Nicoli, J. R., Uzeda, M. & Vieira, L. Q. (2001). Implantation of bacteria from human pulpal necrosis and translocation from root canals in gnotobiotic mice. J Endod 27, 605–609.[Medline]
Ribeiro Sobrinho, A. P., Maltos, S. M., Farias, L. M., Carvalho, M. A. R., Nicoli, J. R., Uzeda, M. & Vieira, L. Q. (2002). Cytokine production in response to endodontic infection in germ-free mice. Oral Microbiol Immunol 17, 344–353.[CrossRef][Medline]
Schwartzman, R. A. & Cidlowski, J. A. (1993). Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14, 133–151.[CrossRef][Medline]
Smith, C. A., Farrah, T. & Goodwin, R. G. (1994). The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76, 959–962.[CrossRef][Medline]
Stevens, D. L. (1997). Superantigens: their role in infectious diseases. Immunol Invest 26, 275–281.[Medline]
Sutter, V. L., Citron, D. M. & Finegold, S. M. (1980). Wadsworth Anaerobic Bacteriology Manual, 2nd edn. St Louis, MO: Mosby Company.
Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462.[Abstract/Free Full Text]
Weinrauch, Y. & Zychlinsky, A. (1999). The induction of apoptosis by bacterial pathogens. Annu Rev Microbiol 53, 155–187.[CrossRef][Medline]
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INTRODUCTION
METHODS
