Periodontal disease is a chronic inflammatory disease where groups of periodontopathic bacteria such as P. gingivalis plays a major role in its initiation and progression. A number of epidemiological studies have suggested that periodontal disease is a risk factor for various systemic diseases and conditions, including cardiovascular disease, type 2 diabetes, NAFLD and rheumatoid arthritis28. Interestingly, obesity increases the risk of these diseases29,30,31. In addition, obesity is also associated with an increased risk of periodontal disease. Therefore, it is possible that periodontal disease and obesity have similar effects on these systemic conditions.

The common systemic effect of obesity and periodontal disease is considered to be low-grade inflammation13. It is well-known that diet-induced obesity is implicated in systemic low grade inflammation. Nutritional fatty acids activate Toll-like receptor 4 (TLR4) signalling in adipocytes and macrophages. In addition, the capacity of fatty acids to induce inflammatory signalling in adipocytes or macrophages is blunted in the absence of TLR432,33. Furthermore, adipose tissue lipolysis from hypertrophied adipocytes, could serve as a naturally occurring ligand for TLR4 to induce inflammation. It is apparent that gut microbial ecology could be an important factor in the development of obesity by affecting energy homeostasis. For example, Cani et al. elegantly demonstrated that the composition of the gut microbiota is influenced by a high-fat diet- and genetically obese ob/ob-induced metabolic endotoxemia34. Components originating from the gut microbiota, such as lipopolysaccharide, lipoteichoic acid, peptidoglycan, flagellin and bacterial DNA can cause immune system activation and subsequent inflammation.

Proposed mechanisms for periodontal diseases inducing systemic inflammation have included: (i) the direct effect of infectious agents or their products and (ii) increased expression of cytokines, chemokines and cell adhesion molecules produced in periodontitis lesions35. Additional mechanisms may include, (iii) translocation of swallowed P. gingivalis from the gut to the circulationg system and (iv) alteration of gut microbial composition-induced increases in gut epithelial permeability by swallowed P. gingivalis. The first hypothesis is based on the lesion size of periodontal disease (periodontitis). It is reported that the mean dentogingival epithelial surface area of periodontitis patients where subgingival biofilm is contacting a thinning and/or ulcerated gingival sulcular epithelium is approximately 20 cm236. This area is considered to act as an entrance of periodontopathic bacteria into the systemic circulation. This mechanism is supported by a number of studies that have demonstrated an association between periodontal disease and endotoxemia. However, periodontal treatment-induced endotoxemia is detectable as early as 5 min after instrumentation and disappears at 30 min37,38. In our study, increases in the serum endotoxin level was observed 1 hr after a single administration of P. gingivalis and peaked at 3 hrs. For the second hypothesis, there is no direct evidence that increased inflammatory markers in periodontitis patients are in fact derived from inflamed periodontal tissues. For the third possibility, although P. gingivalis was detected in the jejunal and ileal contents and colonic contents up to 3 hrs and 16 hrs after a single administration, respectively, it was not detected in the blood of P. ginigivalis-administered mice whereas other bacterial DNA was detected in the blood. Therefore, it is highly likely that detected endotoxins are not derived from P. gingivalis.

Given that recent findings have implicated an altered gut microbiota as a contributor of not only metabolic diseases such as atherosclerosis39,40, type 2 diabetes41 and NAFLD42 but also immune diseases such as rheumatoid arthritis43 that are also associated with periodontitis, it is reasonable to assume that the systemic inflammatory changes seen in P. gingivalis-administered mice could be attributable to this altered gut microbiota. Furthermore, periodontal diseases themselves could be modulated by alteration of gut microbiota-induced systemic inflammation. In support of this hypothesis, it has been reported that high-fat diet-induced obesity increased alveolar bone resorption, a characteristic feature of periodontitis44,45.

The swallowed saliva of periodontitis patients is reported to contain up to 109 bacteria/ml, in 1.0–1.5 L/day46,47,48, making a total of greater than 1012 bacteria/day. Since the bacterial flora of the oral cavity is distinct from that of the gut49, it is possible that swallowed bacteria could affect the composition of the gut microflora. In fact, it has been reported that oral probiotic intervention alters gut bacterial composition50. Thus, it is unlikely that oral bacteria alone could be causative agents of endotoxemia in periodontitis patients.

In the present study, oral administration of P. gingivalis induced a change of bacterial composition in the ileal microflora. Although several studies have shown the beneficial effect of Bacteroidetes phylum on the gut51,52, we observed an increased proportion of the order Bacteroidales belonging Bacteroidetes phylum, accompanied by insulin resistance and the alteration of gene expression in adipose tissue and liver. The expression of several proinflammatory genes and anti-inflammatory or insulin sensitivity-improving genes were upregulated or downregulated, respectively, in the adipose tissue and the liver of P. gingivalis-administered mice. In addition, expression of Sirt1 which is reported to induce an increase in glucose uptake and insulin signalling was also downregulated. It has also been reported that SIRT1 expression is inversely related to inflammatory gene expression, particularly TNF-α. These changes in proinflammatory and anti-inflammatory gene expression may contribute to increases in serum glucose levels and in insulin intolerance. Although the effect of P. gingivalis is not robust, continuous deterioration of glucose metabolism could have significant effect.

One of the possible reasons for the discrepancy of inflammation-associated change of microbiota between previous studies and our study could be the difference of the site from where the samples were obtained. Previous studies analyzed faecal or caecal samples, whereas we analysed ileal contents. The ileum is considered to be an important organ because chylomicron is formed into small vesicles in the epithelial cells of the ileum and lipid is mainly absorbed from the ileum. In addition, Peyer's Patches are located in the ileal wall. Therefore, as previously demonstrated with caecal bacterial53, the bacterial composition in the ileum may have significant impact on systemic inflammation. It is also noteworthy that in humans, Bacteroidetes may promote type 2 diabetes through an endotoxin-induced inflammatory response54.

Henao-Mejia et al., demonstrated that a significant expansion of Porphyrmonadaceae was found following HFD or with methionine-choline-deficient diet administration in the faecal microbiota in the inflammasome-deficient setting and was associated with progression of NAFLD and obesity55. We propose that administered P. gingivalis is not directly responsible for the increase of the “family” Porphyromonadaceae in the gut, as we did not detect P. gingivalis in the gut by using specific primers. However, bacteria belonging to this “family” of bacteria may play a role in the induction of endotoxemia and subsequent inflammatory responses. Alternatively, P. gingivalis may suppress inflammasome activation by other bacteria. For example, it has been reported that P. gingivalis suppresses inflammasome activity through inhibition of endocytosis56.

Another interesting finding from this study was that mRNA expression of alkaline phosphatase was downregulated in the ileum of P. gingivalis-administered mice. Kalannen et al. previously demonstrated that a defect in intestinal alkaline phosphatase (IAP), was associated with high-fat diet-induced metabolic syndrome and endogenous and orally supplemented IAP inhibited endotoxin absorption, as well as reversed metabolic syndrome in mice57. Therefore, IAP is considered to play an important role in the suppression of endotoxemia. Although the underlying mechanisms by which oral administration of P. gingivalis and/or alteration of gut microbiota downregulate the mRNA of alkaline phosphatase has not been clarified, downregulation of the Akp3 gene may be a factor for elevated systemic inflammation. Moreover, gene expression analysis of the small intestine demonstrated downregulated mRNA expression of the tight junction protein ZO-1 in P. gingivalis-administered mice. In previous studies on mouse models, the endotoxemia following high-fat diet administration was associated with reduced expression of genes encoding for ZO-1 and occludin34. These results suggest that administered P. gingivalis alters the gut microbiota and alters the gut epithelial cell barrier function, resulting in increased gut permeability. However, the mechanism for how this change in the gut microbiota impairs gut barrier function has not been elucidated. P. gingivalis administration-induced alterations of the gut microbiota also induced upregulated mRNA expression of various proinflammatory cytokines. It is not known whether these inflammatory changes of the large intestine affect systemic inflammatory responses.

In conclusion, in the present study, we demonstrated that oral administration of P. gingivalis induced alteration of gut microbiota as well as inflammatory changes in various tissues and organs. These changes are considered to be attributable to increases in levels of endotoxin in the blood. However, as with previous observations on the influence of high-fat diet-induced metabolic endotoxemia induced changes of the gut microbiota, it remains to be elucidated whether a cause-and-effect relationship exists between oral administration of P. gingivalis-induced systemic inflammation and changes in the gut microbiota. Also, further investigations are needed to examine whether other oral bacteria have similar effects on the systemic metabolism. Because the composition of the oral microflora and gut microflora are quite distinct, the considerable flow of large quantities of oral bacteria into gut during the frequent act of swallowing could disturb the balance of the gut microflora. Furthermore, bacterial components responsible for the alteration of gut bacterial composition have also not been elucidated. Therefore, further studies are needed to clarify the exact mechanisms of how P. gingivalis induces shifts in the gut microbiota towards the production of metabolic products and shifts in the composition of bacterial species responsible for the induction of metabolic syndrome.