Dysbiotic oral microbiota contributes to alveolar bone loss associated with obesity in mice

Abstract Periodontal diseases (PD) are inflammatory conditions that affect the teeth supporting tissues. Increased body fat tissues may contribute to activation of the systemic inflammatory response, leading to comorbidities. Some studies have shown that individuals with obesity present higher incidence of PD than eutrophics. Objective To investigate the impact of obesity on periodontal tissues and oral microbiota in mice. Methodology Two obesity mice models were performed, one using 12 weeks of the dietary protocol with a high-fat (HF) diet in C57BL/6 mice and the other using leptin receptor-deficient mice (db/db-/-), which became spontaneously obese. After euthanasia, a DNA-DNA hybridization technique was employed to evaluate the microbiota composition and topical application of chlorhexidine (CHX), an antiseptic, was used to investigate the impact of the oral microbiota on the alveolar bone regarding obesity. Results Increased adipose tissue may induce alveolar bone loss, neutrophil recruitment, and changes in the oral biofilm, similar to that observed in an experimental model of PD. Topical application of CHX impaired bone changes. Conclusion Obesity may induce changes in the oral microbiota and neutrophil recruitment, which are associated with alveolar bone loss.


Introduction
Periodontal diseases (PD) are inflammatory conditions characterized by damage of the teeth supporting tissues. The dysbiosis of the oral microbiota is a determining factor in the pathogenesis of PD.¹ Aggregatibacter actinomycetemcomitans (Aa), a Gramnegative bacterium, is an important microorganism related to PD, acting as a key participant in oral dysbiosis. 2 Studies also show that systemic conditions, such as obesity, diabetes and rheumatoid arthritis impact PD. 3,4,5 Thus, patients with obesity, for example, are at a higher risk of developing PD. 3 Obesity is an abnormal or excessive fat accumulation that can impair health. 6 White adipose tissue is an endocrine organ related to the synthesis of compounds such as adipocytokines, associated to energy homeostasis 7 and to immune response regulation. 8 Diverse evidence supports endocrine regulation of bone metabolism by adipose tissue. 9 In mice, Montalvany-Antonucci, et al. 10 (2018) showed harmful effects of HF diet on bone microarchitecture. 10 However, the mechanisms that underlie the association between PD and obesity are deficiently understood.
Obesity causes dysbiosis and, on the contrary, a dysbiotic microbiota may favor obesity. 11,12 In a dysbiotic context, some microorganisms can interfere in the physiological functions of the immune system, and, in an oral environment it may contribute to PD progression.¹ Obesity may affect periodontal tissues by causing an exacerbated systemic inflammatory response. 13 Our hypothesis is that the systemic inflammation due to obesity induces oral dysbiosis, which contribute to alveolar bone loss. Therefore, we evaluated the oral microbiota in two models of obesity in mice and whether dysbiosis contributed to alveolar bone loss observed in obese mice.

Methodology
Mice C57BL6/J wild-type (WT), specific pathogen-free mice, were obtained from the animal facility of the Federal University of Minas Gerais (Universidade Federal de Minas Gerais, UFMG, Brazil), and leptinreceptor deficient mice (db/db -/-) were obtained from the Immunopharmacology laboratory facilities (UFMG, Brazil). The mice were housed in separate cages, under standard conditions and with free access to food and water. All animals were from 6 to 8 weeks old and were separated according to gender. Depending on the experiment, 4, 5, or  for 5 min, at room temperature) and the pellet was suspended in Phosphate-Buffered Saline (PBS) to obtain an inoculum with 1 × 10 9 CFU/mL. For PD induction, as described by Madeira, et al. 16 (2012), 100 μL of inoculum plus 1.5% carboxymethylcellulose was placed in the mice's oral cavity, using a micropipette. The sham-infected mice were administered 100 uL of PBS with 1.5% carboxymethylcellulose in the oral cavity.
The protocol was repeated after 48 and 96 h.

Quantification of alveolar bone loss
The evaluation of alveolar bone loss was performed as previously described. 16 The maxillae were hemisected, exposed to 15% hydrogen peroxide overnight, and mechanically defleshed. Then, they were stained with 0.3% methylene blue. The palatal faces of the molars were photographed with 20× magnification using a stereomicroscope (Metrimpex Hungary/PZO, Labimex, Hungary) and a digital camera (Kodak EasyShare C743, Rochester, USA). The images were analyzed using the Image J software (National Institutes of Health, USA).
Quantitative analysis was used to measure the area between the Cement-Enamel Junction (CEJ) and the Alveolar Bone Crest (ABC) in the first upper right molar.
All samples were evaluated in a blinded manner by a single examiner (IMC).

Myeloperoxidase concentration
The activity of myeloperoxidase (MPO) in the mice's periodontal tissues was measured as previously described. 16 After euthanasia, hemimaxillae, including teeth, periodontal soft tissues, and alveolar bone were removed and processed. Subsequently, the samples were assayed and MPO activity was measured by changes in Optical Density (OD) at 450 nm, using tetramethylbenzidine (1.6 mM) and H 2 O 2 (0.5 mM).
The results were expressed as MPO activity by 100 mg of tissue.

Enzyme-Linked Immunosorbent Assay (ELISA)
The adipocytokine concentrations were measured in the mice's maxillae and/or serum. For protein extraction, the palatal periodontal tissue was homogenized in PBS containing anti-proteases  t-test were performed. Grouped analysis was made by means of Two-way ANOVA followed by Bonferroni.

B a c t e r i a l q u a n t i f i c a t i o n by D N A-D N A hybridization
The data were analyzed using GraphPad Prism 8 (GraphPad Inc., San Diego, CA, USA). p-values <0.05 were considered statistically significant.

Results
High-fat (HF) diet induces body weight gain, increased adiposity and higher leptin production in mice.  The grouped analysis was performed by means of two-way ANOVA followed by Bonferroni in the body weight analysis. One-way ANOVA was followed by Newman-Keuls. The Student's t-test was performed when two groups were analyzed

High-fat diet induces oral dysbiosis
Oral microbiota dysbiosis is a crucial feature in the pathogenesis of PD. Thus, the load of bacterial species in the oral microbiota was evaluated by means of DNA-DNA hybridization ( Figure 3C). Mice infected with Aa presented a lower amount of Actinomyces odontolyticus when compared to non-infected mice fed with both types of diet ( Figure 3C). Also, mice infected with Aa presented lower Eubacterium saburreum levels ( Figure 3C), when compared to non-infected mice fed with standard diet. Mice fed with the HF diet also presented increased levels of two Fusobacterium nucleatum subspecies (Prevotella nigrescens, and Nisseria mucosa) when compared to the groups fed with the control diet ( Figure 3C). Altogether, these results indicate an oral dysbiosis profile associated with HF diet consumption. Heat-map representing differences on the oral microbiota analysis between mice fed with the control diet infected or non-infected with Aa, and non-infected mice fed with the HF diet. Groups: C -Sham-infected group on the control diet. Aa -Group on the control diet, infected with A. actinomycetemcomitans HF -Group on the HF diet, non-infected. N=5. *p<0.05 Aa compared to HF; #p<0.05 C compared to Aa; +p<0.05 C compared to HF. A two-way ANOVA -Bonferroni multiple comparison test was performed. The values (mean ± S.E.M) are representative of two independent experiments J Appl Oral Sci.

2022;30:e20220238 7/11
Topical application of chlorhexidine impaired HF diet-induced alveolar bone loss and changes in alveolar bone microarchitecture We performed an oral topical application of CHX, an antimicrobial agent with a broad anti-microbial action spectrum on both Gram-positive and Gram-negative bacteria. The use of CHX impaired HF diet-induced alveolar bone loss (Figures 4A and 4B). Moreover, the HF diet was associated with increased MPO activity in periodontal tissues, which was also impaired by CHX application ( Figure 4C). CHX application did not affect the increased HF diet-induced adiposity ( Figure 4D).
The results of the micro-CT analysis showed that HF diet consumption changed alveolar bone architecture ( Figure 5A). HF diet consumption induced a significant reduction in Bone Mineral Density (BMD) when compared to the control diet ( Figure 5B). The mice on the HF diet, which received topical application of CHX, showed less bone mineral density loss when compared to the HF diet group ( Figure 5B). The results also showed a negative correlation between BMD and the adiposity index in the HF group (r=−0.9601) ( Figure 5C). Regardless of the topical application of CHX, the HF diet induced: i) increased percentage ratio of bone volume related to total sample volume ( Figure 5D), and ii) decreased trabecular thickness  For example, a deep sequencing analysis of the oral microbiota is suitable to obtain a more in-depth profile of the oral microbiota composition, mainly during dysbiosis. Moreover, despite the use of in vivo studies, including gene knockout mice, suitable for detailed mechanisms, we did not sufficiently evaluate some inflammatory markers such as cytokines in this study.

Conclusion
HF diet-induced oral dysbiosis and subsequent alveolar bone loss. CHX use impaired bone changes and neutrophil influx but had no effect on adiposity gain.

Conflict of interest
The authors have declared no conflicts of interest in this study.

Data availability statement
All data generated or analyzed during this study are included in this published article.