Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans. These diseases include neoplastic, autoimmune, and chronic inflammatory diseases1. The etiology of many chronic diseases remains unclear but is understood to be complex and multifactorial, involving both genetic predisposition and the introduction of environmental factors. While the perpetuators of inflammation remain elusive, the cellular and molecular profiles of immune activation overlap considerably with those patterns observed in host responses to pathogens2.
Mounting evidence implicates infection with microbial pathogens in the development and progression of chronic inflammation and its diverse clinical manifestations2,3. Pathogens can induce and sustain chronic inflammation directly by subverting the host immune system and establishing persistent infections4. In the absence of microbial persistence, infection can precipitate chronic inflammation from autoimmune reactions triggered by molecular mimicry to self-antigens, changes in self-antigens that render them immunogenic, or damage that releases previously masked host antigens. Rarely however have specific pathogens been identified as the universal cause of a particular chronic disease. Rather, the majority of available data suggests that pathogens use distinct mechanisms to elicit chronic inflammation with a wide spectrum of clinical manifestations and disease outcomes in the genetically susceptible host3. Thus, a detailed understanding of the mechanisms by which specific pathogens induce chronic inflammation may have major implications for public health, as well as treatment and prevention of many chronic diseases.
Although the host and pathogen specific mechanisms contributing to the induction and maintenance of chronic inflammation are poorly understood, advances in modeling of pathogen-induced chronic inflammation have begun to further our understanding of these processes. The P. gingivalis oral infection model is a unique, well-characterized mouse model of pathogen-induced chronic inflammation that permits the analysis of host and pathogen specific mechanisms contributing to chronic inflammation at local (oral bone loss) and systemic sites (atherosclerosis)5,6.
P. gingivalis is a Gram-negative, anaerobic oral pathogen implicated in human periodontal disease, an infection-driven chronic inflammatory disease characterized by the destruction of tooth supporting tissue7. In addition to pathology at the initial site of infection, accumulating evidence implicates P. gingivalis-induced chronic inflammation in the development and progression of systemic diseases including atherosclerosis5, a disease characterized by chronic inflammation of the arterial vessel wall. Oral infection of specific-pathogen free mice with P. gingivalis induces a local inflammatory response that results in destruction of tooth supporting alveolar bone8. P. gingivalis can be recovered from the mouths of infected mice up to 42 days post-infection8 and mice develop high levels of pathogen-specific serum antibody titers9. In an established mouse model of atherosclerosis using Apolipoprotein-E-/- mice (ApoE-/-), oral infection with P. gingivalis induces chronic inflammation that drives inflammatory plaque deposition within the aortic sinus10 and the innominate artery11. Progressive inflammation within the innominate artery of P. gingivalis-infected mice can be monitored in live animals using in vivo MRI. Histologically, arterial lesions from P. gingivalis-infected mice exhibit increased accumulation of lipids accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators12. Use of this model in knockout mice has elucidated the role of host signaling components and inflammatory mediators, as well as the cell specific interactions that drive P. gingivalis-induced immunopathology12–14. In addition, experiments utilizing defined bacterial mutants have identified critical P. gingivalis virulence factors contributing to chronic inflammation at local and systemic sites15.
This article details methodologies for the assessment of P. gingivalis-induced chronic inflammation at local and systemic sites. We provide a detailed protocol for the analysis of alveolar bone loss by microCT using Amira software. In addition, we define the utility of serial in vivo live animal MRI for the assessment of progressive inflammation within the innominate artery. We include methodologies for the visualization and quantification of inflammatory plaque in arterial lesions, and describe their histological characterization. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
1. Growth and Cultivation of Bacteria
2. Oral Infection
NOTE: As illustrated in Figure 1, using the appropriate mouse model and oral infection regimen, P. gingivalis induces chronic inflammation and immunopathology at local (oral cavity) and systemic sites (arteries).
3. Micro-computed Tomography (microCT)
4. Assessment of Atherosclerosis
5. Histological Assessment of Atherosclerotic Lesions
6. Immunohistochemical Characterization of Atherosclerotic Lesions.
The steps below outline a general antibody-based protocol routinely employed to assess atherosclerotic lesions in P. gingivalis-infected mice. This protocol requires optimization for each antibody or reagent.
7. MRI
Using the appropriate mouse model and oral infection regimen, P. gingivalis induces chronic inflammation and immunopathology at local (oral cavity) and systemic sites (arteries) (Figure 1).
In mice, oral infection with P. gingivalis induces a local inflammatory response that drives the destruction of tooth supporting alveolar bone8. P. gingivalis-infected mice develop serum antibody responses to this organism that are predominantly of the IgG isotype9. Results shown in Figure 4 are representative of an experiment in which C57BL/6 were infected with P.gingivalis three times at two day intervals and sacrificed 6 weeks later for the assessment of alveolar bone loss by microCT. Volumetric analysis using Amira software reveals that P. gingivalis-infected mice exhibit significant bone loss compared with uninfected controls (Figure 4A). Visual inspection of reconstructed hemi-maxillae illustrates an increase in exposed surface area of the molar roots in infected mice as compared to controls (Figure 4B and Figure 4C).
In atherosclerosis-prone ApoE-/- mice, P. gingivalis induces chronic inflammation that drives alveolar bone loss12 and inflammatory plaque deposition within the aortic sinus15 and innominate artery11. P. gingivalis-induced atherosclerosis occurs as early as 24 hrs following the last infection and can be prevented by immunization prior to infection16. Progressive inflammation in the innominate artery of P. gingivalis-infected mice can be monitored in live mice by serial in vivo MRI at various time points post-infection (Figure 5). En face measurements of Sudan IV stained aortas demonstrates that P. gingivalis infection significantly increases lipid deposition and lesional area on the intimal surface (Figure 6). At the time of sacrifice, histology and immunohistochemistry can be used to qualitatively or quantitatively characterize atherosclerotic lesions in the context of cellular composition, the expression of various antigens, and lipid content. Immunohistochemical analysis of aortic sinus lesions reveals increased macrophage infiltration and elevated expression of the innate immune receptor Toll-like receptor 2 (TLR2) in P. gingivalis-infected mice (Figure 7).
Figure 1. P. gingivalis-induced chronic inflammation at local and systemic sites. Prior to infection with P. gingivalis mice are administered antibiotics ad libitum in their drinking water for 10-14 days followed by a two-day antibiotic rest period. Antibiotic treatment suppresses the indigenous oral flora and facilitates colonization. For the induction of alveolar loss, mice are infected three times at two-day intervals and alveolar bone volume is measured 6 weeks post-infection. When assessing atherosclerosis, atherosclerosis-prone ApoE-/- mice are typically infected 5 times a week for 3 weeks and sacrificed 16-24 weeks post-infection. Progressive inflammation within the innominate artery of live mice can be measured by serial in vivo MRI at various time points post-infection. Histology and immunohistochemistry can be used to stain for lipids and inflammatory cells at termination of the experiment to validate imaging data. At the time of sacrifice, alveolar bone loss is measured by microCT and global atherosclerotic burden is assessed by en face staining of whole aortas.
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Figure 2. Mouse hemi-maxilla illustrating the positioning of OBS 1 and OBS 2. The three molars are labeled (M1-M3) and relevant anatomically terminology is indicated.
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Figure 3. Mouse hemi-maxilla illustrating the positioning of OBS 1 through 5.
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Figure 4. Alveolar bone lossas measured by microCT. Male C57BL/6 were orally infected with P. gingivalis or vehicle alone (uninfected) and alveolar bone volume was evaluated by microCT 6 weeks later using Amira. (A) Alveolar bone volume in hemi-maxillae from uninfected and P. gingivalis-infected C57BL/6. The results represent bone volume above the reference plane (120 microns from the CEJ). Data are expressed as bone volume SD from n=8 mice per group. *** p<0.001, compared to uninfected controls. (B) and (C) Representative 3D reconstructions of hemi-maxillae from uninfected (B) and P. gingivalis-infected (C) mice. A significant decrease in alveolar bone volume can be seen in P. gingivalis-infected mice when compared to uninfected controls. Arrowheads indicate areas where visible bone loss occurs in P. gingivalis-infected mice (note the increase in exposed surface area of the molar roots).
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Figure 5. Progressive inflammation within the innominate artery following P. gingivalis infection as measured by MRI. (A) Representative Magnetic Resonance (MR) angiogram of aortic arch and major vessels of an ApoE-/- mouse. (B) Axial MR image from the yellow line in A of the innominate artery of a mouse, 0.3mm below its bifurcation. Innominate arteries were imaged by MRA at baseline and at 12 and 16 wk post-infection. (C) The temporal change in luminal area (mm2) was calculated for individual mice (n = 10-12/group). Uninfected ApoE-/- (blue); P. gingivalis-infected ApoE-/- (red).
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Figure 6. En face determination of lesional areain the whole aorta. (A) Sudan IV staining of aorta en face lesions 16 wk post-infection with P. gingivalis. (B) Quantification of lipid content within the total aorta of uninfected (white symbols) and P. gingivalis-infected mice (black symbols) (n = 10–13/group). Percentage of aorta occupied by lipids was calculated using ImageJ. *p < 0.05.
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Figure 7. Immunohistochemical analysis of aortic sinus lesions. Male ApoE-/- mice fed a normal chow diet were infected with P. gingivalis or uninfected and sacrificed 16 weeks post-infection. Cryosections obtained from the aortic sinus were stained with anti-mouse F4/80 and TLR2. Scale bar, 100 μm.
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The P. gingivalis oral infection model provides a valuable tool for the study of pathogen-induced chronic inflammation at local and systemic sites. This unique model permits the characterization of both host and pathogen specific mechanisms contributing to chronic inflammation and immunopathology. In addition, the model can be used to screen for novel therapeutic strategies, including immunization and pharmacological intervention. The steps outlined in this protocol describe the successful use of this model and detail methodologies to assess the initiation, progression, and outcome of P. gingivalis-induced chronic inflammation.
There are several critical aspects to keep in mind when using this protocol to examine inflammatory bone loss. First, it should be noted that the outcome of infection with P. gingivalis is determined by three key factors: 1) genetic susceptibility of the host to infection 2) pathogen virulence (genetics of the pathogen) and 3) the resulting host-pathogen interaction (interaction of the two genomes). Susceptibility to P. gingivalis-induced alveolar bone loss is genetically determined in mice, thus care must be taken when selecting the strain of mice for study17. Differential host responses among inbred strains of mice can be taken advantage of to conduct forward genetic screens and characterize genes involved in susceptible and resistance to pathogen-induced chronic inflammation. In addition, considerable heterogeneity exists in the ability of different P. gingivalis strains to induce alveolar bone loss in mice18. This protocol uses mice on the C57BL/6 background because of the availability of transgenic mice and their susceptibility to atherosclerosis. P. gingivalis strain 381 induces alveolar bone loss and atherosclerosis in mice on the C57BL/6 background, and several bacterial mutants have been engineered using this strain.
Mice are particularly resistant to atherosclerosis and the development of overt arterial lesions requires the use of genetically modified mouse models of atherosclerosis. We use the ApoE-/- mouse model because it is well-established mouse model of atherosclerosis, does not require feeding of high fat diet for lesion formation, and recapitulates many aspects of human disease19. The type of diet to feed the animals during the course of the experiment is an important variable. For the majority of our work, we feed mice a normal chow diet to avoid the contribution of exogenous lipids in the interpretation of our results. In preliminary studies, we found that feeding mice a high fat diet masks differences in en face lesion area between uninfected and P. gingivalis-infected mice within the aortic sinus. However, high fat diet and P. gingivalis infection work synergistically when the progression of inflammation is monitored in the innominate by MRI or histology11. In mice, the innominate artery has a high degree of lesion progression, and lesions in this artery express features characteristic of clinical disease in humans including vessel narrowing, atrophic media, perivascular inflammation, and plaque disruption. Distinctions in the cellular composition of lesions are evident at different anatomical sites. Macrophages are the primary immune cells infiltrating aortic sinus lesions, while innominate artery lesions are composed of both macrophages and T cells.
Experimental duration and the time point at which inflammatory endpoints are evaluated are additional factors to consider when assessing initiation, progression, and outcome of P. gingivalis-induced atherosclerosis. We previously demonstrated that P. gingivalis-infected ApoE-/- mice exhibit macrophage infiltration, elevated expression of innate immune markers, and increased deposition of inflammatory plaque as early as 24h following the last infection within the aortic sinus and this can be prevented by immunization16. In our hands, inflammation and immunopathology increase with advancing age and are evident up to 24 weeks post-infection. However, decisions regarding duration of the study ultimately rely on the underlying hypothesis being studied, the mode of analysis and prior knowledge of the extent of atherosclerosis under specific environmental conditions.
The use of non-invasive imaging techniques to monitor progressive inflammation in the innominate artery can be used to guide experimental duration. Serial MRI allows for detailed studies of atherosclerosis progression in the same animal that can depict the narrowing of the arterial lumen and small vessel wall areas20. In contrast to traditional methods, such as lipid staining of dissected vessels, MR imaging does not require euthanasia and allows for longitudinal studies to assess the initiation and progression of atherosclerosis. In conjunction with transgenic mice, bacterial mutants, or experimental treatments, the temporal information provided by MRI can be used to evaluate the effect of host genetics, pathogen virulence factors, and therapeutic intervention. As an added benefit, histology and immunohistochemistry can be used to stain for lipids and inflammatory cells at termination of the experiment to validate imaging data. We recently used these methods to demonstrate that oral infection with P. gingivalis accelerates atherosclerosis in the innominate arterties of ApoE-/- mice, that immunization provides protection from plaque progression, and correlates with decreases in the accumulation of lipids and inflammatory cells11.
In summary, this protocol outlines the steps required to produce a robust model of pathogen-induced chronic inflammation, as well as the methods used to assess inflammation at local and systemic sites. Aside from using this model to examine host and pathogen specific mechanisms involved in inflammatory bone loss and atherosclerosis, it can be adapted to study the contribution of pathogen-induced chronic inflammation to additional disease models. This can be accomplished by using transgenic mouse models of disease including rheumatoid arthritis, diabetes, and cancer. Emerging evidence indicates that a number of chronic diseases of unknown etiology may have infectious origins. These diseases include neoplastic, autoimmune, and inflammatory disease, and together compromise the major causes of morbidity and mortality worldwide. Thus, the use of animal models to examine the role of pathogens in diseases driven by chronic inflammation has the potential for broad therapeutic impact and improved diagnostics.
The authors have nothing to disclose.
This work was supported by National Institutes of Allergy and Infectious Diseases Grant P01 A1078894 to C.A.G.
Name of the Material/Eqiupment | Company | Catalogue Number | Comments |
Amira analysis software | Visualization Sciences Group | ||
Anaerobic chamber DW Scientific Model MG500 | Microbiology International | ||
BHI | Becton-Dickinson | 211059 | |
Hemin | Sigma-Aldrich | 51280-5G | |
Menadione (Vitamin K) | Sigma-Aldrich | M5625-25G | |
Yeast Extract | Becton-Dickinson | 212750 | |
Carboxymethyl cellulose (medium viscocity) | Sigma-Aldrich | C-4888 | |
Sulfamethoxazole and Trimethoprim Oral Suspension 200 mg/40 mg per 5ml | Hi-Tech Pharmacal | NDC 50383-823-16 | |
μCT 40 | Scanco | ||
HistoChoice Tissue Fixative | Sigma-Aldrich | H2904 | |
Sudan IV | Sigma-Aldrich | S4261-25G | |
vertical-bore 11.7T Avance spectrometer | Bruker | ||
Paravision | Paravision | ||
ImageJ | NIH | ||
rat anti-mouse F4/80 | Serotec | MCA497R | |
rat anti-mouse TLR2 | eBioscience | 13-9021-80 | |
Leica S4 Dissecting Scope | Leica | ||
Microm HM 550 Cryostat | Microm |