Periodontitis, which affects up to 10% of people in severe cases, is an immune-related disease caused by the interaction between oral bacteria and immune cells in biofilm, resulting in periodontal tissue damage (Nicu et al., 2009; Gonzales, 2015; Herrera et al., 2022). Since periodontitis is the result of local immune imbalance, many systemic disorders, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), have been found to be associated with periodontitis (Sete et al., 2016). In recent years, gastrointestinal disorders related to immunity, like inflammatory bowel disease (IBD), have also been reported to be related to periodontitis (Kitamoto et al., 2020b; Sete et al., 2016; Zhou et al., 2021).

Oral and gastrointestinal mucosa is continuous with each other, and both are associated with microbiology and immunology (Kitamoto et al., 2020b). Firmicutes and Bacteroidetes, which make up over 90% of the entire population, are the two main dominating microbiota in the human digestive tract, and subdominant microbiota includes Proteobacteria, Actinobacteria, and Verrucomicrobia, which regulate physiology, metabolism, immunity, and health-disease processes (Vieira et al., 2016; Fan and Pedersen, 2021). Interestingly, the human oral is made up of six major germ classes, including Actinobacteria, Bacteroides, Firmicutes, Fusobacteria, Proteobacteria, along with Spirochetes (Xu et al., 2015). When periodontitis occurs in the oral cavity, it results in the expansion of the Klebsiella/Enterobacter species, which can be transferred to the lower digestive tract and colonized (Kitamoto et al., 2020b; Magne et al., 2020).

The result of chronic intestinal inflammation depends on the makeup of the gut microbiota, immunological response, host genetic factors, and how these variables interact. Cross-talk between the gut microbiota and the host immune system can either prevent or mitigate this condition (Cao, 2017). Studies have shown that colonization of the gut by oral bacteria can be engaged in the progression of intestinal inflammatory diseases by interfering with intestinal flora composition and intestinal immunity (Cao, 2017; Kitamoto et al., 2020b). Oral microorganisms can mediate intestinal inflammation by invading the intestinal epithelium, causing the release of inflammatory factors, decreasing the killing capacity of macrophages and natural killer (NK) cells, and promoting pro-inflammatory Th 1 and Th 17 responses (Kitamoto et al., 2020b; Zhou et al., 2019; Read et al., 2021), and there is growing evidence showed that in the pathophysiology of gastrointestinal illnesses, the “oral-intestinal axis” might be crucial (Kitamoto et al., 2020b). However, fewer studies have been conducted to survey the impact of microecological changes in gut flora on periodontitis progression, establishing a brand-new avenue for periodontitis clinical therapy.

Currently, it has been hypothesized that the progression of colitis and periodontitis may be influenced by the invasion of CD4⁺T cells (Zhang et al., 2021). It is widely accepted that immune cell infiltration of mucosal tissues is a hallmark of periodontitis. T cells are its main immune cell population, which is related to the expression of inflammatory cytokines (Gonzales, 2015; Williams et al., 2021). In periodontitis, dendritic cells(DCs) capture and process antigens and express the costimulatory molecules and cytokines needed for antigen presentation to B and T cells. DCs also play an important role in the “tolerance” of T cells to autoantigens, thereby reducing the risk of autoimmune reactions. Meanwhile, DCs are an effective stimulant for NK cells. NK cells, a unique subgroup of cytotoxic T lymphocytes, are abundant in periodontitis lesions, and NK cell activation has a causal relationship with periodontal tissue destruction (Krämer et al., 2013; Meyle and Chapple, 2015). Moreover, bacterial invasion of periodontal tissue leads to CD4⁺ T cells that differentiate into Th17 cells, which protect our body against pathogens by producing mucosal immune response and bone damage (Tsukasaki et al., 2018). Thus, T cells may be the bridge between intestinal inflammation and periodontitis. However, the immune response of T cells may be influenced by the nutritional microenvironment, such as oxygen, glucose, and microbial metabolites (Ramsay and Cantrell, 2015). The immune system’s metabolism can be influenced by metabolites produced by the intestinal microbiota, with TMAO, short-chain fatty acids (SCFAs), and bile acids (BAs) being particularly significant. (Michaudel and Sokol, 2020). TMAO, a toxin generated from gut bacteria and derived from nutrients such as dietary choline, has been found to be associated with immunological disorders. For instance, TMAO raises the risk of death from chronic kidney disease, nonalcoholic fatty liver disease, and colorectal cancer (Wang et al., 2020a; Fan and Pedersen, 2021; Zhang et al., 2022). Studies have shown that long-term TMAO supplementation can cause significant changes in the microbial composition of the entire cecum leading to the activation of the intestinal immune system and chronic inflammatory illnesses progression (Zhu et al., 2016; Atarashi et al., 2017). Simultaneously, clinical findings in patients with periodontitis are often accompanied by increased circulating levels of TMAO (Xiao et al., 2021; Zhou et al., 2022). Accordingly, TMAO may play an essential role in periodontitis and intestinal inflammation progression. On the one hand, by activating NF-κB, boosting the expression of inflammatory genes (IL-1), as well as enhancing oxidative stress, TMAO could enhance the indication of inflammatory markers (Yang et al., 2019). On the other hand, TMAO has also been correlated with the improved efficacy of immunotherapy in the clinical cohort (Wang et al., 2022b). TMAO enhances M1 macrophage polarization through NLRP3 inflammasome activation, and polarized macrophages participate in the effect on periodontal osteoclast by TGF-β (Jiang et al., 2019; Wu et al., 2020; Wu et al., 2021).

Up to now, no researchers have focused on the role of TMAO in the relationship between intestinal inflammation and oral inflammation. The widespread presence of TMAO in daily red meat is a growing concern not only for individual health but also for the health of the planet. Therefore, we designed a series of follow-up experiments to prove that TMAO can affect the process of periodontitis. In this study, we used DMB, a structural analog of choline, which nonlethally reduces the TMAO concentration in mice (Wang et al., 2015). By administering exogenous TMAO and its inhibitor DMB to mice, we observed the changes in periodontal tissue, intestinal tissue, and intestinal flora in mice with different periodontitis. Further proved that TMAO affected the development of periodontitis by involving the regulation of the intestinal-oral axis.

TMAO, a pro-inflammatory metabolite, has been found to be involved in inflammation, inflammation-related immunity, and other mechanisms in diseases such as IBD (Hosseinkhani et al., 2021; Zhou et al., 2022). The intestinal tract promotes the production of ILC3 by IL-2 regulatory Treg cells through the microflora and IL-1β dependent axis, thereby maintaining intestinal immunity (Zhou et al., 2019). But the colonization resistance of intestinal microbiota can be destroyed by TMAO, allowing oral microorganisms to invade and disrupt the intestinal barrier, thus promoting intestinal inflammation (Atarashi et al., 2017; Kitamoto et al., 2020b; Zhu et al., 2016). Consistent with these reports, by analyzing the intestinal barrier through morphological assessment of intestinal villi, we discovered that the intestinal barrier of the P+TMAO group was severely compromised, as evidenced by the group’s shorter intestinal villi and deeper intestinal crypts.

Firmicutes and Bacteroidetes comprise more than 90% of the entire bacterial community, plus the subdominant phyla Proteobacteria, Actinobacteria, and Verrucomicrobia constitute the vast majority of the human intestinal microbiota (Magne et al., 2020). Recently, dietary choline has been linked to changes in intestinal bacteria and even intestinal ecological problems, including major alterations in the abundance, diversity, metabolism, and function of microorganisms (Koay et al., 2021). Reduced variety is typically a sign of an unbalanced gut microbiota, with an increase in pathogenic bacteria and a decrease in helpful bacteria (Li et al., 2021a). As the choline metabolites, TMAO usually comes from L-carnitine, phosphatidylcholine, and choline, which are found in red meat, fish, eggs, as well as high-fat dairy products. Global analyses of community structure revealed distinctions between chow-fed (0.07% choline) and those fed high choline (1.0%) diets. These differences were more obvious in the presence of DMB. Studies have shown that aging will increase TMAO levels in peripheral and central tissues, induce intestinal microbiota imbalance and decrease α-diversity, while DMB can effectively reduce TMAO levels and improve peripheral metabolism (Wang et al., 2015; Lanz et al., 2022). This is consistent with our experimental results that TMAO can change the structure of intestinal flora and even cause intestinal flora imbalance. Intestinal flora imbalance is also associated with a variety of pathological conditions, for example, the digestive tract, including irritable bowel syndrome and diarrhea; the immune system, including multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis; the central nervous system, including Parkinson’s disease and Alzheimer’s disease and autism, as well as host energy metabolism including type 2 diabetes, obesity, and atherosclerosis (Magne et al., 2020).

Although it remains unclear whether gut flora dysbiosis is a begetter or a result of the above diseases, the ratio of phylum Firmicutes/Bacteroidetes (F/B), which expresses how the two dominant phyla are related to one another, has been demonstrated to be linked to several pathological conditions (Magne et al., 2020). The average ratio of F/B of humans is 2.6. An increased F/B ratio has been identified as the marker of a pro-inflammatory state and immune imbalance in autoimmune diseases (Chen et al., 2021a; Yang et al., 2015; Magne et al., 2020), such as obesity, diabetes, and cardiovascular disease. Moreover, the study found a difference in the major group F/B ratio and the gut microbial composition between obese and lean individuals (Wang et al., 2020b). The entire constituent of the gut microbiota is altered in obese patients, and studies have found an increased F/B ratio in hereditary obese mice and mice with high-fat diets (de Sant'Ana et al., 2019). Jun L et al. proposed that TMAO concentrations were associated with the overall microbial compositions. The abundance of 10 bacterial species was significantly correlated with plasma TMAO concentration, including 8 Firmicutes species, one Bacteroidetes species, and one Actinobacteria species (Li et al., 2022). Consistent with previous findings, we found an increase in the abundance of the Firmicutes phylum in the P+TMAO group, indicating that TMAO could influence the intestinal microenvironment by affecting the formation and abundance of intestinal microorganisms.

Similar to the findings of Wang X. et al., the decreased abundance of Firmicutes was positively connected with the TMAO level in our study (Wang et al., 2020a). Interestingly, we discovered a noticeable reduction in the richness of Firmicutes and an abundance of Proteobacteria increased in the P+DMB group. DMB affects the liver and intestinal metabolism by decreasing choline levels, which in turn affects the structure and function of intestinal flora, such as decreasing the abundance of Firmicutes (Zeisel and da Costa, 2009; Trebicka et al., 2021). Choline is critical in synthesizing acetylcholine, methylation, and gene expression of liver and muscle function (Arias et al., 2020). Even if endogenous choline synthesis is possible, it is insufficient to suit human demands. Many diseases related to intestinal microorganisms, such as atherosclerosis, nonalcoholic fatty liver disease, lipoprotein secretion, and even nervous system problems, are considered to be affected by choline deficiency (Zeisel and da Costa, 2009). Furthermore, Proteobacteria are closely associated with hepatic and intestinal metabolism. For example, the abundance of Proteobacteria is positively correlated with nonalcoholic fatty liver fibrosis in the normal body mass index population (Jasirwan et al., 2021).

Although the increase in the prevalence of phylum Proteobacteria is a sign of an unstable microbial community (dysbiosis) and a possible diagnostic standard of diseases, in a healthy and stable state, the relative abundance of Proteobacteria in the intestine sometimes climbs to 45% (average: 2.5% over 15 months) without showing any symptoms of sickness (Shin et al., 2015). Additionally, under certain conditions, some Enterobacteriaceae family species and genera, including Fusobacteriumvarium, Staphylococcus, and Porphyromonas gingivalis, are linked to oral pathology, may ectopically colonize the intestine (Kitamoto et al., 2020a). However, when our findings were compared with previous studies, we found a correlation between TMAO and periodontal immunity. Clinical and animal studies have found that periodontitis is often accompanied by increased TMAO levels in peripheral blood (Xiao et al., 2021; Zhou et al., 2022). TMAO has been reported to trigger activated NLRP3 inflammatory corpuscles in a dose-dependent and time-dependent manner to induce vascular inflammation, endothelial dysfunction, and ROS production (Yue et al., 2017; Wu et al., 2020). The NLRP3/caspase-1 signaling pathway is engaged in the IL-1β production in M1 macrophages in vivo. NLRP3 inhibitors could significantly reverse this signaling pathway’s activation and improve the symptoms of periodontitis in rats (Sun et al., 2021). In addition, another research showed that TMAO levels were positively correlated with the expression level of IL-1β and hsCRP (Chou et al., 2019). Activated macrophages and lymphocytes produce IL-1β, which is necessary for T cell activation and proliferation, and cooperate with IL-6 to cause periodontal tissue damage and promote periodontitis (Beklen et al., 2007). Similarly, TGF-β not only controls T cell differentiation into regulatory and effector subsets, which is important for the growth and maintenance of immune cells, but it also promotes the production of IL-1, IL-6, as well as TGF-β via inflammatory cells, which helps to the onset of periodontitis (Travis and Sheppard, 2014; Leite et al., 2015). Clinical experiments have shown that TGF-β and IL-1β are highly expressed in the gingival tissue of patients with chronic severe periodontitis (Beklen et al., 2007; Chen et al., 2021b). We discovered that in P+TMAO and P groups, IL-1β and TGF-β were significantly higher than in other groups, accompanied by periodontal tissue damage, which is consistent with previous observations. Notably, our results did not rule out the association of TMAO with other factors, for instance, anti-inflammatory cytokines (IL-10) in the pathogenesis of periodontitis. On the contrary, TMAO may work synergistically with various factors to promote the development of periodontitis.

Recent studies have shown the intimate connection between bone metabolism and intestinal microbiota (Sjögren et al., 2012; Yan et al., 2016; Guss et al., 2017). Changes in the intestinal microflora are related to many diseases that lead to bone loss, including malnutrition, IBD, obesity, and metabolic disease (Guss et al., 2017). Animal experiments demonstrated that TMAO activates the ROS-dependent NF-κB signaling pathway to enhance osteoclast polarization and cause bone loss in mice (Li et al., 2019; Wang et al., 2022a). Additionally, TMAO can induce impaired intestinal barrier function, resulting in elevated levels of IL-1β and TGF-β (Nanto-Hara et al., 2020; Li et al., 2021b; Xie et al., 2022; Zhang et al., 2022). Elevated IL-1β and TGF-β circulate to periodontal tissues and damage them. Besides, TGF-β could involve in osteoclastic effects through TLR4/NF-κB signaling (Jiang et al., 2019; Wu et al., 2021). Based on the Micro CT results, our results agree with earlier studies.

In conclusion, as shown in Figure 4 , our data imply that intestinal inflammation caused by the ecological imbalance of intestinal flora due to exogenous TMAO is related to the progression of periodontitis by involving the regulation of the intestinal-oral axis. However, medical treatment represented by DMB results in the decrease of TMAO level, which can effectively delay the progression of periodontitis and open up a new way for the treatment of periodontitis in the future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA916071 .

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Jilin University.

QW and YS contributed equally to this work. QW and YS conceived and designed the experiments. QW, TZ and CJ conducted the data and bioinformatics analyses. YS, WX and LA conducted the animal experiments. QW, YS, WX and LA wrote the manuscripts. LA and WX oversaw the completion of this study and edited the manuscript. All authors contributed to the article and approved the submitted version.