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Impact of the Oral Commensal Flora on Alveolar Bone Homeostasis K. Irie, C.M. Novince and R.P. Darveau J DENT RES 2014 93: 801 originally published online 16 June 2014 DOI: 10.1177/0022034514540173 The online version of this article can be found at: http://jdr.sagepub.com/content/93/8/801
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research-article2014
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Research Reports Biological
K. Irie1,2,3†, C.M. Novince1†, and R.P. Darveau1* 1
Department of Periodontics, School of Dentistry, University of Washington, Seattle, WA 98195, USA; 2Department of Preventive Dentistry and Dental Public Health, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi, 464-8650, Japan; and 3Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan; †authors contributing equally to this work; *corresponding author, [email protected]
Impact of the Oral Commensal Flora on Alveolar Bone Homeostasis
J Dent Res 93(8):801-806, 2014
Abstract Homeostasis of healthy periodontal tissues is affected by innate and adaptive immunosurveillance mechanisms in response to the normal oral flora. Recent comparisons of germ-free (GF) and normal specific-pathogen-free (SPF) mice have revealed the impact of host immunosurveillance mechanisms in response to the normal oral flora on alveolar bone height. Prior reports that alveolar bone height is significantly less in normal SPF mice compared with their age- and strain-matched GF counterparts suggest that naturally occurring alveolar bone loss is a normal component of healthy periodontal tissue homeostasis. In this report, histomorphometric analyses confirmed increased alveolar bone loss and revealed increased numbers of TRAP+ osteoclastic cells lining the alveolar bone surface in SPF compared with GF mice. Increased numbers of RANKL+ cells and IL17+ cells in the periodontium of SPF mice demonstrate possible molecular mechanisms mediating the up-regulated osteoclastogenesis and alveolar bone loss in SPF mice compared with GF mice. Increased numbers of T-lymphocytic cells and T-helper cells in the junctional epithelium of SPF mice compared with GF mice suggest that the adaptive immune response contributes to physiologic alveolar bone loss in the healthy periodontium. This GF animal model study notably begins to elucidate the impact of host immunosurveillance mechanisms in response to the normal oral flora, mediating catabolic alveolar bone homeostasis in the healthy periodontium.
KEY WORDS:
helper T-cells, RANKL protein, interleukin-17, tartrate-resistant acid phosphatase, osteoclastic bone loss, periodontium.
DOI: 10.1177/0022034514540173 Received March 5, 2014; Last revision May 27, 2014; Accepted May 28, 2014 © International & American Associations for Dental Research
Introduction
R
ecently, it has been reported that naturally occurring alveolar bone loss is significantly blunted in germ-free (GF) mice when compared with specific-pathogen-free (SPF) mice (Hajishengallis et al., 2011). Evidence that the normal oral flora were responsible for the observed alveolar bone loss in SPF mice was obtained by both co-caging experiments of GF and SPF mice as well as longitudinal measurements of natural bone loss during the first 15 wk of life (Hajishengallis et al., 2011). These observations are consistent with those of an earlier study (Brown et al., 1969) and suggest that the commensal oral flora affects alveolar bone homeostasis in the healthy periodontium. While it has been shown that host immune response mechanisms to perio-pathogenic bacteria mediate pathologic alveolar bone loss (Kornman et al., 1997; Baker, 2000; Taubman et al., 2005), host innate defense mechanisms regulating the normal oral flora and the impact on alveolar bone homeostasis during health are not clear. Periodontal disease–associated alveolar bone loss is driven by the elevated expression of pro-inflammatory signaling factors supporting osteoclastogenesis, resulting in catabolic bone destruction (Di Benedetto et al., 2013; Ebersole et al., 2013). The host immune response to perio-pathogenic bacteria significantly contributes to alveolar bone loss (Kornman et al., 1997; Baker, 2000; Taubman et al., 2005), which is largely attributed to overexpression of receptor activator of nuclear factor-κB (RANKL) on activated T-cells (Taubman et al., 2005; Kawai et al., 2006). Prior studies of gingival tissue harvested from SPF mice vs. GF mice have revealed increased basal expression of pro-inflammatory cytokines (Dixon et al., 2004), including RANKL mRNA and interleukin-17 (IL-17) mRNA (Hajishengallis et al., 2011), which suggests that host innate defense system regulation of the commensal flora may have catabolic effects on alveolar bone homeostasis during health. Since perio-pathogenic inoculated oral flora vs. normal oral flora differs in the quantity and diversity of bacteria, it is unclear whether alveolar bone loss in health is mediated by the same mechanisms driving alveolar bone loss during periodontal disease states. This investigation applies histomorphometric analysis of the linear distance from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) to examine alveolar bone loss in GF vs. SPF mice. We performed immunohistochemistry (IHC) labeling and staining of mouse junctional epithelial (JE) tissues to further investigate the impact of the commensal oral flora on the innate defense system of the periodontium and to begin to
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elucidate host immune response mechanisms having catabolic effects on alveolar bone homeostasis. SPF mice had a more severe alveolar bone loss phenotype, with increased numbers of tartrate-resistant acid phosphatase (TRAP)+ osteoclastic cells and RANKL+ cells at the alveolar bone surface, as well as significantly increased numbers of neutrophils, CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL-17+ cells in the JE tissue. Similar to the up-regulated numbers of osteoclastic and inflammatory/immune cells and elevated expression of proinflammatory cytokines found in the periodontium of periodontitisafflicted mice vs. normal mice (Graves et al., 2012), this study reveals similar relative differences in SPF mice vs. GF mice. These findings imply that the host innate defense response mediating homeostasis with the commensal oral microbiota induces a low-grade basal inflammation which causes alveolar bone loss during health.
J Dent Res 93(8) 2014 Histomorphometry Hematoxylin and eosin (H&E) staining was performed in all specimens. We carried out qualitative analysis to evaluate tissue-cell morphological differences and performed histomorphometric analysis of the linear distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) to quantify alveolar bone loss. Linear measurements were performed at 12 pre-determined sites (every fifth serial section) through the mesio-distal width of the second molars, quantifying the linear distance between the CEJ and ABC, at both the buccal and lingual/palatal surfaces. Measurements were performed via a micro-grid at 200X magnification (Irie et al., 2008) with a Nikon Eclipse E400 light microscope (Nikon Corp., Tokyo, Japan). Representative images (100X) were acquired via a Spot RT camera (SPOT Imaging Solutions, Sterling Heights, MI, USA) and MetaVue software (Molecular Devices LLC, Sunnyvale, CA, USA).
Materials & Methods Mice
Immunohistochemistry
Animal procedures described in this study were approved by the institutional animal care and use committees, in compliance with established Federal and state policies. Germ-free (GF) C3H/Orl mice (Charles River Laboratories International, Wilmington, MA, USA) were maintained in isolators under sterile conditions at the Royal Veterinary College, University of London. GF mice were fed a soft chow diet. The sterility of GF animals was examined by aerobic and anaerobic culture of oral swabs and fecal pellets on non-selective media and by polymerase chain reaction (PCR) with universal 16S primers. Specific-pathogen-free (SPF) mice were maintained in individually ventilated cages at the animal care facilities of Queen Mary University of London. SPF mice were fed a standard chow diet. Original GF mice were divided, and half were raised in conventional cages to become the SPF mice; GF and SPF mice were propagated under uniform breeding conditions. The bacterial composition of the natural oral flora of the C3H/ Orl SPF mice has been previously characterized (Hajishengallis et al., 2011).
We performed immunohistochemistry (IHC) labeling for primary antibodies in second molar sections to evaluate the expression of neutrophils, T-cell populations, and pro-inflammatory signaling factors within the JE tissue and adjacent alveolar bone surface. IHC primary antibody staining and analyses were performed in intermittent serial sections, at 12 pre-determined sites (every fifth serial section) through the mesio-distal width of the second molars. Approximately 12 sections per second molar specimen were stained for each primary antibody, which included neutrophil elastase (sc-71674; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD3 (ab5690; Abcam, Cambridge, MA, USA), CD4 (sc-71674; Santa Cruz Biotechnology), IL-17 (ab79056; Abcam), RANKL (ab9957; Abcam), or an IgG matched isotype control. No primary antibody controls were performed for each antibody of interest. Standard IHC staining protocol was performed in conjunction with Abcam IHC-P staining kits. Neutrophil elastase+ (neutrophil) cells, CD3+ (T-lymphocytic) cells, CD4+ (T-helper) cells, and IL-17+ cells were enumerated per JE tissue area. RANKL+ cells occurring at the edge of the alveolar bone surface were enumerated and reported as cells per millimeter (Sanbe et al., 2009). Representative images (200X) of the JE tissue were acquired at the buccal surface. In addition, we carried out tartrate-resistant acid phosphatase (TRAP) staining to enumerate TRAP+ bone lining osteoclastic cells per millimeter bone perimeter at the edge of the alveolar bone surface (Sanbe et al., 2009). TRAP+ cells were detected by the azo dye method (Sigma-Aldrich Kit, St. Louis, MO, USA), and Mayer’s hematoxylin counterstaining was used for labeling cell nuclei. Representative images (200X) of the alveolar bone surface were acquired at the buccal surface.
Histology Eleven- to 12-week-old GF mice and SPF mice (n = 6/gp) were euthanized, and tissues were harvested for histological processing. Upper (maxilla) and lower (mandible) jaws were dissected, and the periodontal soft tissues were not disturbed. Jaws were immediately fixed in Bouin`s solution for 24 hr, rinsed with 70% ethanol, and demineralized in acetic acid/formalin/sodium chloride solution for 2 wk. Specimens were embedded in paraffin in a bucco-lingual orientation. Serial frontal sections (5 μm) were cut parallel to the long axis of each tooth (sagittal), yielding sections for analyses from the mesial/mesio-buccal root of the second molar through the distal/disto-buccal root of the second molar. Two serial sections were mounted per charged glass slides. Sections were numbered, and slides were maintained in corresponding order. All quantitative analyses were performed by a single blinded examiner (K.I.).
Statistical Analysis Initial experiments revealed no significant differences in the number of positive cells of interest per junctional epithelium tissue area between the JE of the mandibular second molar vs.
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J Dent Res 93(8) 2014 803 Impact of Oral Commensal Flora on Alveolar Bone Homeostasis the maxillary second molar, within both the GF group and the SPF group (p < .01, random mixed-model test). Therefore, enumeration of positive cells of interest per JE tissue area was performed in either maxillary second molar sections or mandibular second molar sections from each mouse. Student`s two-tailed unpaired t test and non-parametric MannWhitney test for comparison between the two groups were performed with SPSS statistical software (SPSS 17.0J for Windows; SPSS Japan, Tokyo, Japan). Data are presented as mean ± standard deviation, and statistical significance is p < .05.
䐡 CEJ JE
䐟
Cementum C ABC AB
䐠
Results Commensal Bacteria Increase Alveolar Bone Loss
Figure 1. Diagram of mouse periodontal tissue for histological analysis. AB, alveolar bone; ABC, alveolar bone crest; CEJ, cemento-enamel junction; JE, junctional epithelium. (1) The distances between CEJ and ABC were measured to evaluate the degree of alveolar bone resorption. (2) RANKL+ cells and TRAP+ osteoclastic cells occurring along the edge of the alveolar bone surface were counted. (3) The number of neutrophils, CD3, CD4, and IL-17positive cells infiltrating the JE area were enumerated.
Linear measurements (Fig. 1) assessing the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) demonstrated significantly greater alveolar bone loss at second molars of SPF mice vs. GF mice. This histomorphometric analysis revealed increased alveolar bone loss in SPF mice vs. GF mice, which was more severe at the lingual/palatal surfaces (roughly 1.5X greater in SPF mice vs. GF mice) than the buccal surfaces (roughly 1.3X greater in SPF mice vs. GF mice) (Figs. 2A, 2B).
Commensal Bacteria Increase Osteoclastic Cell Numbers Lining Alveolar Bone Based on the exacerbated alveolar bone loss phenotype of the SPF mice vs. GF mice, we enumerated osteoclastic cells lining the alveolar bone surface. Histomorphometric analysis demonstrated increased numbers of TRAP+ osteoclastic cells per millimeter bone perimeter at the edge of the alveolar bone surface in SPF mice vs. GF mice (Figs. 2C, 2D), which correlates with the increased alveolar bone loss found in SPF mice vs. GF mice.
Commensal Bacteria Increase T-cell Numbers, IL-17, and RANKL Expression As has been reported previously (Tsukamoto et al., 2012; Zenobia et al., 2013), there were significantly more neutrophils located in the JE of SPF mice vs. GF mice (Figs. 3A, 3C). To begin to elucidate oral commensal flora-driven host immune response mechanisms having catabolic effects on alveolar bone homeostasis during health, we assessed T-cell numbers, IL-17, and RANKL expression in the periodontium of SPF mice vs. GF mice. IHC demonstrated increased CD3+ (T-lymphocytic) cell numbers, CD4+ (T-helper) cell numbers, and IL-17+ cell numbers in the JE tissue of SPF mice vs. GF mice (Figs. 3B, 3C). IHC labeling for
the osteoclastogenic signaling factor RANKL revealed significantly increased RANKL+ cell numbers along the edge of the alveolar bone surface in SPF mice vs. GF mice (Figs. 3D, 3E).
Discussion In 1969, Brown and co-authors published the first manuscript reporting increased alveolar bone loss in SPF mice compared with GF mice (Brown et al., 1969). In that early report, the loss of alveolar bone height in SPF mice was interpreted as further evidence for the contribution of bacteria to periodontitis-associated pathologic bone loss (Brown et al., 1969). More recently, a second report of alveolar bone loss in untreated SPF compared with GF mice was published in an investigation elucidating virulence mechanisms of P. gingivalis (Hajishengallis et al., 2011). Two separate and independent approaches validated that SPF mice compared with GF mice have increased alveolar bone loss, and demonstrated that commensal colonization of the mouse was necessary for observation of the decrease in alveolar bone height. In contrast to Brown and co-authors’ (1969) explanation of increased alveolar bone loss in SPF mice in the context of periodontal disease, Hajishengallis and co-authors (2011) and the present study interpreted the increased alveolar bone loss as a consequence of normal host homeostasis in health. Notably, and not previously reported, SPF mice vs. GF mice have increased numbers of TRAP+ osteoclastic cells lining the alveolar bone surface, which correlates with the increased alveolar bone loss phenotype of the SPF mice. The increased neutrophils, CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL17+ cells in the JE tissue and increased RANKL+ cells at
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J Dent Res 93(8) 2014
fluid isolated from individuals with periodontitis vs. periodontally healthy individuals (Vernal et al., 2005). The decreased CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL17+ cells in the JE tissue and decreased RANKL+ cells at the alveolar bone surface in GF mice are novel findings in the periodontium of the GF animal model. While activated T-cells are recognized as the primary cell population expressing supra-physiological levels of RANKL (Kong et al., 1999; Kotake et al., 2001; Weitzmann et al., 2001) and IL-17 (Kotake et al., 1999; Sato et al., 2006; Adamopoulos and Bowman, 2008) under inflammatory disease states resulting in pathologic bone loss, recent investigations reporting that activated neutrophils express RANKL (Poubelle et al., 2007; Chakravarti et al., 2009) and IL-17 (Hoshino et al., 2008; Li et al., 2010; Lin Figure 2. Histomorphometric analysis of alveolar bone loss. (A, B) Measures of the linear et al., 2011; Keijsers et al., 2013) suggest distance between the CEJ and ABC and (C, D) TRAP+ osteoclastic cells per mm alveolar bone that both innate and adaptive immune surface in GF mice vs. SPF mice (*p < .01; n = 6). Arrows in C indicate TRAP+ osteoclastic cells contribute to alveolar bone loss. cells lining the alveolar bone surface. ABC, alveolar bone crest; CEJ, cemento-enamel junction; Additionally, stromal/osteoblastic cells GF, germ-free mice; SPF, specific-pathogen-free mice. are known to up-regulate RANKL expression during periodontal disease states associated with catabolic alveolar the alveolar bone surface of SPF mice suggest that the host bone loss (Teitelbaum, 2000; Boyle et al., 2003). While not immune response mechanisms mediating homeostasis with the within the scope of this study, future investigations elucidating commensal oral microbiota induce a basal low-grade inflammathe RANKL and IL-17 expression fold increase within specific tory state supporting osteoclastogenesis, resulting in naturally periodontal cell populations, and the relative contributions occurring alveolar bone loss in the healthy periodontium. across cell populations, will clarify the cellular-molecular mechAdvances in osteoimmunology research demonstrating that anisms mediating alveolar bone loss in the periodontium during hematopoietic cells regulate osteoblastogenesis highlight the health and disease. need for future investigations assessing potential alterations in Page and Schroeder (1982) postulated that the subgingival bone formation in SPF vs. GF mice, which could contribute to plaque range of effectiveness in generating alveolar bone loss is the increased alveolar bone loss phenotype of the SPF mice. about 2.5 mm, which was driven by Garant and Cho’s (1979) Evaluation of findings from this investigation of the impact of theory that locally produced bone resorption factors have an commensal bacteria on healthy periodontium vs. studies assessing effective radius of action, and by findings that the distance from the impact of perio-pathogenic bacteria on diseased periodontium the apical extension of the subgingival plaque to the alveolar provides indirect evidence that host immune response mechacrest ranged from 0.5 mm to 2.7 mm (Waerhaug, 1979). When nisms intended to maintain homeostasis with the oral flora medione considers the proposed range of effectiveness of subgingival ate alveolar bone loss in both health and disease. Similar to plaque in causing pathologic alveolar bone loss, the host increased CD3+ T-lymphocytic and CD4+ T-helper cell numbers immune response to the normal oral flora may play a role in found infiltrating periodontitis-afflicted vs. healthy gingival tissue establishing the physiologic dimensions of biologic width (Celenligil et al., 1990), SPF mice vs. GF mice had significantly (Gargiulo et al., 1961). The biologic width, composed of the increased numbers of CD3+ T-lymphocytic and CD4+ T-helper junctional epithelium and supra-crestal connective tissue surcells infiltrating the JE tissue. Furthermore, the four-fold- and rounding teeth, has been reported to range from 1.5 mm to two-fold-increased CD3+ T-lymphocytic cell numbers and 2.7 mm in healthy periodontium (Schmidt et al., 2013). While RANKL expression in the periodontal tissue of SPF mice vs. GF future investigations are necessary, oral commensal flora intermice closely parallel the reported 4-fold- and 2.5-fold-elevated actions with the host immune defense system may support T-lymphocytic cell numbers and RANKL expression in periodonbiologic width tissue homeostasis, providing for a protective titis-afflicted vs. healthy gingival tissue (Kawai et al., 2006). Last, zone of junctional epithelium and supra-crestal connective tisthe increased IL-17+ cell numbers found in the JE of SPF mice sue in periodontal health. vs. GF mice correlate with elevated IL-17 expression reported in While 11- to 12-week-old mice were studied for the evaluasupernatants of gingival tissue cell cultures and gingival crevicular tion of early alterations in alveolar bone homeostasis in the
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J Dent Res 93(8) 2014 805 Impact of Oral Commensal Flora on Alveolar Bone Homeostasis mature young adult skeleton, further investigations of growing GF mice and aging adult GF mice are needed to elucidate the impact of the normal oral flora on the development and aging of the alveolar bone. We acknowledge that findings reported here in the C3H/Orl GF mice may not be consistent across mouse strains, because genetic differences in mouse strains have implications for commensal flora composition, host immune response, and skeletal tissue physiology. Future investigations of the commensal oral flora composition, junctional epithelial immune cells, and alveolar bone homeostasis in the periodontium of different GF mouse strains will begin to reveal the role of genetics in the commensal flora interactions with the innate defense system of the periodontium and its impact on naturally occurring alveolar bone loss. The non-uniform hardness diets administered to the SPF mice vs. Figure 3. Immunohistochemical examination of JE tissue and adjacent alveolar bone surface. (A) Neutrophil elastase staining demonstrating neutrophil infiltration into the JE tissue. (B) IL-17 GF mice, a potential study design weakstaining demonstrating IL-17+ cell infiltration into the JE tissue. (C) Enumeration of neutrophils ness, will need to be evaluated, since no and lymphocytic (CD3+, CD4+, IL-17+) cells per JE tissue area in GF mice vs. SPF mice (*p known prior studies have investigated the < .01; n = 6). (D, E) RANKL+ cells per mm alveolar bone surface in GF mice vs. SPF mice (*p impact of diet hardness on alveolar bone < .01; n = 6). Arrows in D indicate RANKL+ cells lining the alveolar bone surface. GF, germhomeostasis in GF mice. free mice; JE, junctional epithelial; SPF, specific-pathogen-free mice. This novel investigation of the impact of the normal oral microbiome on the host immune defense system in the healthy periodontium begins to elucidate immune regulatory in the normal periodontium supports osteoclast-mediated alveomechanisms having catabolic effects on alveolar bone homeolar bone loss during health. Future investigations characterizing stasis during health. This is the first known histomorphometric differences in host immune defense regulation of the normal study evaluating the alveolar bone loss phenotype in GF vs. SPF oral microbiome in healthy periodontium vs. perio-pathogenic mice, which was assessed previously by morphometric analyses oral flora in diseased periodontium will advance our underof defleshed maxillae/mandibles. To our knowledge, the enustanding of the mechanisms mediating alveolar bone loss during meration of osteoclastic cell numbers lining the alveolar bone health vs. disease. surface performed here is the initial comparative analysis of osteoclast cell numbers in the alveolar bone of GF mice vs. SPF Acknowledgments mice. Most notably, this is the first known investigation of periodontal tissue homeostasis in the GF animal model for quantitaThis work was supported by National Institutes of Health (NIH) tive assessment of T-cell (CD3+ T-lymphocytic cell and CD4+ grant DE018274 to Richard Darveau. The authors declare no T-helper cell) numbers and IL-17 and RANKL expression in the potential conflicts of interest with respect to the authorship and/ periodontium. Novel findings from this study suggest that the or publication of this article. normal commensal flora stimulates the innate defense system of the periodontium, up-regulating not only the number of neutrophils but also T-cell (CD3+ T-lymphocytic cell and CD4+ References T-helper cell) and IL-17 expression in the JE tissue and RANKL Adamopoulos IE, Bowman EP (2008). Immune regulation of bone loss by expression at the alveolar bone surface. Similar to increased Th17 cells. Arthritis Res Ther 10:225-234. neutrophil numbers infiltrating the JE tissue of SPF mice, we Baker PJ (2000). The role of immune responses in bone loss during perispeculate that the increased T-cell (CD3+ T-lymphocytic cell odontal disease. Microbes Infect 2:1181-1192. and CD4+ T-helper cell) numbers and IL-17 and RANKL Boyle WJ, Simonet WS, Lacey DL (2003). Osteoclast differentiation and expression are due to the host innate defense system inflammaactivation. Nature 423:337-342. 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Impact of the Oral Commensal Flora on Alveolar Bone Homeostasis K. Irie, C.M. Novince and R.P. Darveau J DENT RES 2014 93: 801 originally published online 16 June 2014 DOI: 10.1177/0022034514540173 The online version of this article can be found at: http://jdr.sagepub.com/content/93/8/801
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research-article2014
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93810.1177/0022034514540173
Research Reports Biological
K. Irie1,2,3†, C.M. Novince1†, and R.P. Darveau1* 1
Department of Periodontics, School of Dentistry, University of Washington, Seattle, WA 98195, USA; 2Department of Preventive Dentistry and Dental Public Health, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi, 464-8650, Japan; and 3Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan; †authors contributing equally to this work; *corresponding author, [email protected]
Impact of the Oral Commensal Flora on Alveolar Bone Homeostasis
J Dent Res 93(8):801-806, 2014
Abstract Homeostasis of healthy periodontal tissues is affected by innate and adaptive immunosurveillance mechanisms in response to the normal oral flora. Recent comparisons of germ-free (GF) and normal specific-pathogen-free (SPF) mice have revealed the impact of host immunosurveillance mechanisms in response to the normal oral flora on alveolar bone height. Prior reports that alveolar bone height is significantly less in normal SPF mice compared with their age- and strain-matched GF counterparts suggest that naturally occurring alveolar bone loss is a normal component of healthy periodontal tissue homeostasis. In this report, histomorphometric analyses confirmed increased alveolar bone loss and revealed increased numbers of TRAP+ osteoclastic cells lining the alveolar bone surface in SPF compared with GF mice. Increased numbers of RANKL+ cells and IL17+ cells in the periodontium of SPF mice demonstrate possible molecular mechanisms mediating the up-regulated osteoclastogenesis and alveolar bone loss in SPF mice compared with GF mice. Increased numbers of T-lymphocytic cells and T-helper cells in the junctional epithelium of SPF mice compared with GF mice suggest that the adaptive immune response contributes to physiologic alveolar bone loss in the healthy periodontium. This GF animal model study notably begins to elucidate the impact of host immunosurveillance mechanisms in response to the normal oral flora, mediating catabolic alveolar bone homeostasis in the healthy periodontium.
KEY WORDS:
helper T-cells, RANKL protein, interleukin-17, tartrate-resistant acid phosphatase, osteoclastic bone loss, periodontium.
DOI: 10.1177/0022034514540173 Received March 5, 2014; Last revision May 27, 2014; Accepted May 28, 2014 © International & American Associations for Dental Research
Introduction
R
ecently, it has been reported that naturally occurring alveolar bone loss is significantly blunted in germ-free (GF) mice when compared with specific-pathogen-free (SPF) mice (Hajishengallis et al., 2011). Evidence that the normal oral flora were responsible for the observed alveolar bone loss in SPF mice was obtained by both co-caging experiments of GF and SPF mice as well as longitudinal measurements of natural bone loss during the first 15 wk of life (Hajishengallis et al., 2011). These observations are consistent with those of an earlier study (Brown et al., 1969) and suggest that the commensal oral flora affects alveolar bone homeostasis in the healthy periodontium. While it has been shown that host immune response mechanisms to perio-pathogenic bacteria mediate pathologic alveolar bone loss (Kornman et al., 1997; Baker, 2000; Taubman et al., 2005), host innate defense mechanisms regulating the normal oral flora and the impact on alveolar bone homeostasis during health are not clear. Periodontal disease–associated alveolar bone loss is driven by the elevated expression of pro-inflammatory signaling factors supporting osteoclastogenesis, resulting in catabolic bone destruction (Di Benedetto et al., 2013; Ebersole et al., 2013). The host immune response to perio-pathogenic bacteria significantly contributes to alveolar bone loss (Kornman et al., 1997; Baker, 2000; Taubman et al., 2005), which is largely attributed to overexpression of receptor activator of nuclear factor-κB (RANKL) on activated T-cells (Taubman et al., 2005; Kawai et al., 2006). Prior studies of gingival tissue harvested from SPF mice vs. GF mice have revealed increased basal expression of pro-inflammatory cytokines (Dixon et al., 2004), including RANKL mRNA and interleukin-17 (IL-17) mRNA (Hajishengallis et al., 2011), which suggests that host innate defense system regulation of the commensal flora may have catabolic effects on alveolar bone homeostasis during health. Since perio-pathogenic inoculated oral flora vs. normal oral flora differs in the quantity and diversity of bacteria, it is unclear whether alveolar bone loss in health is mediated by the same mechanisms driving alveolar bone loss during periodontal disease states. This investigation applies histomorphometric analysis of the linear distance from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) to examine alveolar bone loss in GF vs. SPF mice. We performed immunohistochemistry (IHC) labeling and staining of mouse junctional epithelial (JE) tissues to further investigate the impact of the commensal oral flora on the innate defense system of the periodontium and to begin to
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elucidate host immune response mechanisms having catabolic effects on alveolar bone homeostasis. SPF mice had a more severe alveolar bone loss phenotype, with increased numbers of tartrate-resistant acid phosphatase (TRAP)+ osteoclastic cells and RANKL+ cells at the alveolar bone surface, as well as significantly increased numbers of neutrophils, CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL-17+ cells in the JE tissue. Similar to the up-regulated numbers of osteoclastic and inflammatory/immune cells and elevated expression of proinflammatory cytokines found in the periodontium of periodontitisafflicted mice vs. normal mice (Graves et al., 2012), this study reveals similar relative differences in SPF mice vs. GF mice. These findings imply that the host innate defense response mediating homeostasis with the commensal oral microbiota induces a low-grade basal inflammation which causes alveolar bone loss during health.
J Dent Res 93(8) 2014 Histomorphometry Hematoxylin and eosin (H&E) staining was performed in all specimens. We carried out qualitative analysis to evaluate tissue-cell morphological differences and performed histomorphometric analysis of the linear distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) to quantify alveolar bone loss. Linear measurements were performed at 12 pre-determined sites (every fifth serial section) through the mesio-distal width of the second molars, quantifying the linear distance between the CEJ and ABC, at both the buccal and lingual/palatal surfaces. Measurements were performed via a micro-grid at 200X magnification (Irie et al., 2008) with a Nikon Eclipse E400 light microscope (Nikon Corp., Tokyo, Japan). Representative images (100X) were acquired via a Spot RT camera (SPOT Imaging Solutions, Sterling Heights, MI, USA) and MetaVue software (Molecular Devices LLC, Sunnyvale, CA, USA).
Materials & Methods Mice
Immunohistochemistry
Animal procedures described in this study were approved by the institutional animal care and use committees, in compliance with established Federal and state policies. Germ-free (GF) C3H/Orl mice (Charles River Laboratories International, Wilmington, MA, USA) were maintained in isolators under sterile conditions at the Royal Veterinary College, University of London. GF mice were fed a soft chow diet. The sterility of GF animals was examined by aerobic and anaerobic culture of oral swabs and fecal pellets on non-selective media and by polymerase chain reaction (PCR) with universal 16S primers. Specific-pathogen-free (SPF) mice were maintained in individually ventilated cages at the animal care facilities of Queen Mary University of London. SPF mice were fed a standard chow diet. Original GF mice were divided, and half were raised in conventional cages to become the SPF mice; GF and SPF mice were propagated under uniform breeding conditions. The bacterial composition of the natural oral flora of the C3H/ Orl SPF mice has been previously characterized (Hajishengallis et al., 2011).
We performed immunohistochemistry (IHC) labeling for primary antibodies in second molar sections to evaluate the expression of neutrophils, T-cell populations, and pro-inflammatory signaling factors within the JE tissue and adjacent alveolar bone surface. IHC primary antibody staining and analyses were performed in intermittent serial sections, at 12 pre-determined sites (every fifth serial section) through the mesio-distal width of the second molars. Approximately 12 sections per second molar specimen were stained for each primary antibody, which included neutrophil elastase (sc-71674; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD3 (ab5690; Abcam, Cambridge, MA, USA), CD4 (sc-71674; Santa Cruz Biotechnology), IL-17 (ab79056; Abcam), RANKL (ab9957; Abcam), or an IgG matched isotype control. No primary antibody controls were performed for each antibody of interest. Standard IHC staining protocol was performed in conjunction with Abcam IHC-P staining kits. Neutrophil elastase+ (neutrophil) cells, CD3+ (T-lymphocytic) cells, CD4+ (T-helper) cells, and IL-17+ cells were enumerated per JE tissue area. RANKL+ cells occurring at the edge of the alveolar bone surface were enumerated and reported as cells per millimeter (Sanbe et al., 2009). Representative images (200X) of the JE tissue were acquired at the buccal surface. In addition, we carried out tartrate-resistant acid phosphatase (TRAP) staining to enumerate TRAP+ bone lining osteoclastic cells per millimeter bone perimeter at the edge of the alveolar bone surface (Sanbe et al., 2009). TRAP+ cells were detected by the azo dye method (Sigma-Aldrich Kit, St. Louis, MO, USA), and Mayer’s hematoxylin counterstaining was used for labeling cell nuclei. Representative images (200X) of the alveolar bone surface were acquired at the buccal surface.
Histology Eleven- to 12-week-old GF mice and SPF mice (n = 6/gp) were euthanized, and tissues were harvested for histological processing. Upper (maxilla) and lower (mandible) jaws were dissected, and the periodontal soft tissues were not disturbed. Jaws were immediately fixed in Bouin`s solution for 24 hr, rinsed with 70% ethanol, and demineralized in acetic acid/formalin/sodium chloride solution for 2 wk. Specimens were embedded in paraffin in a bucco-lingual orientation. Serial frontal sections (5 μm) were cut parallel to the long axis of each tooth (sagittal), yielding sections for analyses from the mesial/mesio-buccal root of the second molar through the distal/disto-buccal root of the second molar. Two serial sections were mounted per charged glass slides. Sections were numbered, and slides were maintained in corresponding order. All quantitative analyses were performed by a single blinded examiner (K.I.).
Statistical Analysis Initial experiments revealed no significant differences in the number of positive cells of interest per junctional epithelium tissue area between the JE of the mandibular second molar vs.
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J Dent Res 93(8) 2014 803 Impact of Oral Commensal Flora on Alveolar Bone Homeostasis the maxillary second molar, within both the GF group and the SPF group (p < .01, random mixed-model test). Therefore, enumeration of positive cells of interest per JE tissue area was performed in either maxillary second molar sections or mandibular second molar sections from each mouse. Student`s two-tailed unpaired t test and non-parametric MannWhitney test for comparison between the two groups were performed with SPSS statistical software (SPSS 17.0J for Windows; SPSS Japan, Tokyo, Japan). Data are presented as mean ± standard deviation, and statistical significance is p < .05.
䐡 CEJ JE
䐟
Cementum C ABC AB
䐠
Results Commensal Bacteria Increase Alveolar Bone Loss
Figure 1. Diagram of mouse periodontal tissue for histological analysis. AB, alveolar bone; ABC, alveolar bone crest; CEJ, cemento-enamel junction; JE, junctional epithelium. (1) The distances between CEJ and ABC were measured to evaluate the degree of alveolar bone resorption. (2) RANKL+ cells and TRAP+ osteoclastic cells occurring along the edge of the alveolar bone surface were counted. (3) The number of neutrophils, CD3, CD4, and IL-17positive cells infiltrating the JE area were enumerated.
Linear measurements (Fig. 1) assessing the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) demonstrated significantly greater alveolar bone loss at second molars of SPF mice vs. GF mice. This histomorphometric analysis revealed increased alveolar bone loss in SPF mice vs. GF mice, which was more severe at the lingual/palatal surfaces (roughly 1.5X greater in SPF mice vs. GF mice) than the buccal surfaces (roughly 1.3X greater in SPF mice vs. GF mice) (Figs. 2A, 2B).
Commensal Bacteria Increase Osteoclastic Cell Numbers Lining Alveolar Bone Based on the exacerbated alveolar bone loss phenotype of the SPF mice vs. GF mice, we enumerated osteoclastic cells lining the alveolar bone surface. Histomorphometric analysis demonstrated increased numbers of TRAP+ osteoclastic cells per millimeter bone perimeter at the edge of the alveolar bone surface in SPF mice vs. GF mice (Figs. 2C, 2D), which correlates with the increased alveolar bone loss found in SPF mice vs. GF mice.
Commensal Bacteria Increase T-cell Numbers, IL-17, and RANKL Expression As has been reported previously (Tsukamoto et al., 2012; Zenobia et al., 2013), there were significantly more neutrophils located in the JE of SPF mice vs. GF mice (Figs. 3A, 3C). To begin to elucidate oral commensal flora-driven host immune response mechanisms having catabolic effects on alveolar bone homeostasis during health, we assessed T-cell numbers, IL-17, and RANKL expression in the periodontium of SPF mice vs. GF mice. IHC demonstrated increased CD3+ (T-lymphocytic) cell numbers, CD4+ (T-helper) cell numbers, and IL-17+ cell numbers in the JE tissue of SPF mice vs. GF mice (Figs. 3B, 3C). IHC labeling for
the osteoclastogenic signaling factor RANKL revealed significantly increased RANKL+ cell numbers along the edge of the alveolar bone surface in SPF mice vs. GF mice (Figs. 3D, 3E).
Discussion In 1969, Brown and co-authors published the first manuscript reporting increased alveolar bone loss in SPF mice compared with GF mice (Brown et al., 1969). In that early report, the loss of alveolar bone height in SPF mice was interpreted as further evidence for the contribution of bacteria to periodontitis-associated pathologic bone loss (Brown et al., 1969). More recently, a second report of alveolar bone loss in untreated SPF compared with GF mice was published in an investigation elucidating virulence mechanisms of P. gingivalis (Hajishengallis et al., 2011). Two separate and independent approaches validated that SPF mice compared with GF mice have increased alveolar bone loss, and demonstrated that commensal colonization of the mouse was necessary for observation of the decrease in alveolar bone height. In contrast to Brown and co-authors’ (1969) explanation of increased alveolar bone loss in SPF mice in the context of periodontal disease, Hajishengallis and co-authors (2011) and the present study interpreted the increased alveolar bone loss as a consequence of normal host homeostasis in health. Notably, and not previously reported, SPF mice vs. GF mice have increased numbers of TRAP+ osteoclastic cells lining the alveolar bone surface, which correlates with the increased alveolar bone loss phenotype of the SPF mice. The increased neutrophils, CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL17+ cells in the JE tissue and increased RANKL+ cells at
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J Dent Res 93(8) 2014
fluid isolated from individuals with periodontitis vs. periodontally healthy individuals (Vernal et al., 2005). The decreased CD3+ T-lymphocytic cells, CD4+ T-helper cells, and IL17+ cells in the JE tissue and decreased RANKL+ cells at the alveolar bone surface in GF mice are novel findings in the periodontium of the GF animal model. While activated T-cells are recognized as the primary cell population expressing supra-physiological levels of RANKL (Kong et al., 1999; Kotake et al., 2001; Weitzmann et al., 2001) and IL-17 (Kotake et al., 1999; Sato et al., 2006; Adamopoulos and Bowman, 2008) under inflammatory disease states resulting in pathologic bone loss, recent investigations reporting that activated neutrophils express RANKL (Poubelle et al., 2007; Chakravarti et al., 2009) and IL-17 (Hoshino et al., 2008; Li et al., 2010; Lin Figure 2. Histomorphometric analysis of alveolar bone loss. (A, B) Measures of the linear et al., 2011; Keijsers et al., 2013) suggest distance between the CEJ and ABC and (C, D) TRAP+ osteoclastic cells per mm alveolar bone that both innate and adaptive immune surface in GF mice vs. SPF mice (*p < .01; n = 6). Arrows in C indicate TRAP+ osteoclastic cells contribute to alveolar bone loss. cells lining the alveolar bone surface. ABC, alveolar bone crest; CEJ, cemento-enamel junction; Additionally, stromal/osteoblastic cells GF, germ-free mice; SPF, specific-pathogen-free mice. are known to up-regulate RANKL expression during periodontal disease states associated with catabolic alveolar the alveolar bone surface of SPF mice suggest that the host bone loss (Teitelbaum, 2000; Boyle et al., 2003). While not immune response mechanisms mediating homeostasis with the within the scope of this study, future investigations elucidating commensal oral microbiota induce a basal low-grade inflammathe RANKL and IL-17 expression fold increase within specific tory state supporting osteoclastogenesis, resulting in naturally periodontal cell populations, and the relative contributions occurring alveolar bone loss in the healthy periodontium. across cell populations, will clarify the cellular-molecular mechAdvances in osteoimmunology research demonstrating that anisms mediating alveolar bone loss in the periodontium during hematopoietic cells regulate osteoblastogenesis highlight the health and disease. need for future investigations assessing potential alterations in Page and Schroeder (1982) postulated that the subgingival bone formation in SPF vs. GF mice, which could contribute to plaque range of effectiveness in generating alveolar bone loss is the increased alveolar bone loss phenotype of the SPF mice. about 2.5 mm, which was driven by Garant and Cho’s (1979) Evaluation of findings from this investigation of the impact of theory that locally produced bone resorption factors have an commensal bacteria on healthy periodontium vs. studies assessing effective radius of action, and by findings that the distance from the impact of perio-pathogenic bacteria on diseased periodontium the apical extension of the subgingival plaque to the alveolar provides indirect evidence that host immune response mechacrest ranged from 0.5 mm to 2.7 mm (Waerhaug, 1979). When nisms intended to maintain homeostasis with the oral flora medione considers the proposed range of effectiveness of subgingival ate alveolar bone loss in both health and disease. Similar to plaque in causing pathologic alveolar bone loss, the host increased CD3+ T-lymphocytic and CD4+ T-helper cell numbers immune response to the normal oral flora may play a role in found infiltrating periodontitis-afflicted vs. healthy gingival tissue establishing the physiologic dimensions of biologic width (Celenligil et al., 1990), SPF mice vs. GF mice had significantly (Gargiulo et al., 1961). The biologic width, composed of the increased numbers of CD3+ T-lymphocytic and CD4+ T-helper junctional epithelium and supra-crestal connective tissue surcells infiltrating the JE tissue. Furthermore, the four-fold- and rounding teeth, has been reported to range from 1.5 mm to two-fold-increased CD3+ T-lymphocytic cell numbers and 2.7 mm in healthy periodontium (Schmidt et al., 2013). While RANKL expression in the periodontal tissue of SPF mice vs. GF future investigations are necessary, oral commensal flora intermice closely parallel the reported 4-fold- and 2.5-fold-elevated actions with the host immune defense system may support T-lymphocytic cell numbers and RANKL expression in periodonbiologic width tissue homeostasis, providing for a protective titis-afflicted vs. healthy gingival tissue (Kawai et al., 2006). Last, zone of junctional epithelium and supra-crestal connective tisthe increased IL-17+ cell numbers found in the JE of SPF mice sue in periodontal health. vs. GF mice correlate with elevated IL-17 expression reported in While 11- to 12-week-old mice were studied for the evaluasupernatants of gingival tissue cell cultures and gingival crevicular tion of early alterations in alveolar bone homeostasis in the
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J Dent Res 93(8) 2014 805 Impact of Oral Commensal Flora on Alveolar Bone Homeostasis mature young adult skeleton, further investigations of growing GF mice and aging adult GF mice are needed to elucidate the impact of the normal oral flora on the development and aging of the alveolar bone. We acknowledge that findings reported here in the C3H/Orl GF mice may not be consistent across mouse strains, because genetic differences in mouse strains have implications for commensal flora composition, host immune response, and skeletal tissue physiology. Future investigations of the commensal oral flora composition, junctional epithelial immune cells, and alveolar bone homeostasis in the periodontium of different GF mouse strains will begin to reveal the role of genetics in the commensal flora interactions with the innate defense system of the periodontium and its impact on naturally occurring alveolar bone loss. The non-uniform hardness diets administered to the SPF mice vs. Figure 3. Immunohistochemical examination of JE tissue and adjacent alveolar bone surface. (A) Neutrophil elastase staining demonstrating neutrophil infiltration into the JE tissue. (B) IL-17 GF mice, a potential study design weakstaining demonstrating IL-17+ cell infiltration into the JE tissue. (C) Enumeration of neutrophils ness, will need to be evaluated, since no and lymphocytic (CD3+, CD4+, IL-17+) cells per JE tissue area in GF mice vs. SPF mice (*p known prior studies have investigated the < .01; n = 6). (D, E) RANKL+ cells per mm alveolar bone surface in GF mice vs. SPF mice (*p impact of diet hardness on alveolar bone < .01; n = 6). Arrows in D indicate RANKL+ cells lining the alveolar bone surface. GF, germhomeostasis in GF mice. free mice; JE, junctional epithelial; SPF, specific-pathogen-free mice. This novel investigation of the impact of the normal oral microbiome on the host immune defense system in the healthy periodontium begins to elucidate immune regulatory in the normal periodontium supports osteoclast-mediated alveomechanisms having catabolic effects on alveolar bone homeolar bone loss during health. Future investigations characterizing stasis during health. This is the first known histomorphometric differences in host immune defense regulation of the normal study evaluating the alveolar bone loss phenotype in GF vs. SPF oral microbiome in healthy periodontium vs. perio-pathogenic mice, which was assessed previously by morphometric analyses oral flora in diseased periodontium will advance our underof defleshed maxillae/mandibles. To our knowledge, the enustanding of the mechanisms mediating alveolar bone loss during meration of osteoclastic cell numbers lining the alveolar bone health vs. disease. surface performed here is the initial comparative analysis of osteoclast cell numbers in the alveolar bone of GF mice vs. SPF Acknowledgments mice. Most notably, this is the first known investigation of periodontal tissue homeostasis in the GF animal model for quantitaThis work was supported by National Institutes of Health (NIH) tive assessment of T-cell (CD3+ T-lymphocytic cell and CD4+ grant DE018274 to Richard Darveau. The authors declare no T-helper cell) numbers and IL-17 and RANKL expression in the potential conflicts of interest with respect to the authorship and/ periodontium. Novel findings from this study suggest that the or publication of this article. normal commensal flora stimulates the innate defense system of the periodontium, up-regulating not only the number of neutrophils but also T-cell (CD3+ T-lymphocytic cell and CD4+ References T-helper cell) and IL-17 expression in the JE tissue and RANKL Adamopoulos IE, Bowman EP (2008). Immune regulation of bone loss by expression at the alveolar bone surface. Similar to increased Th17 cells. Arthritis Res Ther 10:225-234. neutrophil numbers infiltrating the JE tissue of SPF mice, we Baker PJ (2000). The role of immune responses in bone loss during perispeculate that the increased T-cell (CD3+ T-lymphocytic cell odontal disease. Microbes Infect 2:1181-1192. and CD4+ T-helper cell) numbers and IL-17 and RANKL Boyle WJ, Simonet WS, Lacey DL (2003). Osteoclast differentiation and expression are due to the host innate defense system inflammaactivation. Nature 423:337-342. 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