Fluoride Exposure Induced Bloom of Erysipelatoclostridium Ramosum Mediates the Exacerbation of Obesity in High-fat-diet Fed Mice


 Background: Fluoride, a necessary mineral element for our health, is widely presented in drinking water and foods. The intake of excessive fluoride showed potential risk to human health. A strong relation between fluoride exposure and obesity has been reported. However, the knowledge on the potential mechanisms on fluoride-induced obesity is still limited.Results: In this work, we showed here that fluoride alone did not induce obesity in normal diet fed mice, whereas, it could trigger exacerbation of obesity in high-fat diet (HFD) fed mice. Fluoride impaired intestinal barrier and activated Toll-like receptor 4 (TLR4) signaling to induce obesity, which was further verified in TLR4-/- mice. Furthermore, fluoride could deteriorate the gut microbiota in HFD mice. The fecal microbiota transplantation from fluoride-induced mice was sufficient to induce obesity, while the exacerbation of obesity by fluoride was blocked upon gut microbiota depletion. The fluoride-induced bloom of Erysipelatoclostridium ramosum belonged to Erysipelotrichaceae was responsible for the exacerbation of obesity. In addition, a potential strategy for prevention of fluoride-induced obesity was proposed by intervention with polysaccharides from Fuzhuan brick tea.Conclusions: Overall, these results provide the first evidence of a comprehensive cross-talk mechanism between fluoride and obesity in HFD fed mice, which is mediated by gut microbiota and intestinal barrier. E. ramosum was identified as a crucial mediator of fluoride-induced obesity, which could be explored as potential target for prevention and treatment of obesity with exciting translational value.


Background
Obesity, a complex disease characterized by a high body mass index (BMI) and excess fat accumulation in adipose etc., has dramatically spread throughout the developed and developing countries in the past few decades due to the obesogenic shifts in nutritional composition, excessive intake of calories and lack of exercise [1,2]. It has become a leading public health problem worldwide, which may increase the risks of numerous comorbidities such as type 2 diabetes (T2DM), nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease [3,4]. In China, the latest national prevalence estimates for 2015-2019 showed 16.4% for obesity and 34.3% for overweight in adults (≥ 18 years), respectively [5]. Although the potential mechanisms for obesity and associated metabolic diseases are still not fully understood, increasing evidence supports that the dysbiosis of gut microbiota plays a central role in the development of various obesity-induced diseases [6,7]. The prevention and treatment of obesity are mainly focused on end organs, such as pancreas, adipose tissue, muscle and brain, due to the involvement in glucose regulation and energy balance in the past decades, whereas, the improvement of intestinal health is considered as one of the most effective therapy for obesity-induced diseases in recent years [8]. Indeed, animal and human studies have shown that the modulation of gut microbiota by anti-obesogenic candidates was contributed to the improvement of the obesity and associated metabolic diseases [8][9][10].
To determine the effects of uoride on obesity, the uoride was added into the drinking water at a concentration of 50 mg/L to HFD fed mice (Fig. S1A). The dosage in this work was chosen according to the previous work [21]. As expected, HFD could signi cantly induce the features of obesity compared with the ND group (Fig. S1). It was observed that uoride remarkably exacerbated the obesity in HFD mice over a 10-week period, including increasing body weight ( Fig. S1B-C), accumulation of fat tissues (Fig. S1D-G), increased plasma levels of triglyceride (TG), total cholesterol (TC) and glucose ( Fig. S1H-J). Furthermore, uoride could increase liver weight and hepatic TG ( Fig. S1K and L). As shown in Fig. S1M, uoride exposure did not affect the food intake (p = 0.985), suggesting that exacerbation of the obesity by uoride was not related to the change of energy intake. The reduced colon length and histological damage in colon tissue were observed ( Fig. S2A-C). Furthermore, the protein expression of Claudin-1 and ZO-1 in colon was down-regulated by uoride compared with that of the ND or HFD group (Fig. S2D-G). Thus, the exacerbation of obesity by uoride might be related to the gut health. In addition, a separate replicate experiment was carried out to further con rm the effect of uoride on the obesity in HFD mice, and similar results were observed as showed in Fig. 1, that uoride could exacerbate the obesity in HFD mice. The uoride could also deteriorate the glucose tolerance by signi cant increase of glucose levels in 0 and 15 min, and increase of aera under the curve (AUC) during the oral glucose tolerance test (OGTT) as shown in Fig. 1K-L. Furthermore, the liver injury was observed after exposure of uoride according to the high level of plasma alanine transaminase (ALT) (Fig. 1N). Thus, uoride treatment could exacerbate the detrimental effect of HFD on features of obesity.
Fluoride drives the intestinal barrier permeability and deteriorates the in ammation in HFD mice As described above, the detrimental effect of uoride might be related to the gut health. Thus, the effect of uoride on the intestinal barrier permeability was investigated in the Experiment 2. Similarly, uoride could reduce the colon length ( Fig. 2A-B), lead to histological damage and in ammation in colon tissue ( Fig. 2C-D), and decrease the mRNA and protein expressions of Claudin-1, MUC1 and ZO-1 in colon evaluated by RT-qPCR and immuno uorescence assay ( Fig. 2E-N). The intestinal barrier permeability would lead to leaky gut, and some of gut microbiota, along with toxins such as LPS, might leak into the bloodstream, which was regarded as an important mechanism for metabolic diseases [29]. In this work, the level of bacteria in blood and plasma LPS concentration were signi cantly increased in the HFD-F group compared with those in the ND and HFD groups ( Fig. 2O-P). The effect of uoride on intestinal barrier function was further evaluated in vivo using a uorescein isothiocyanate (FITC)-dextran-based intestinal permeability assay (Fig. 2Q) and in vitro using Caco-2 cell model (Fig. S3). It was found that plasma FITC-dextran level in the HFD-F group was signi cantly higher than that in the HFD group (p =0.038). Moreover, uoride derived Caco-2 cell permeability in vitro evaluated by decreasing transepithelial electrical resistance (TEER). Thus, the experiments in vitro and in vivo both demonstrated that uoride could drive the intestinal barrier permeability. After leaking into the bloodstream, the bacteria and LPS could activate the TLR4, and thereby leading to metabolic in ammation and obesity [15]. Thus, the plasma levels of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and IL-1b and their relative mRNA expression levels (Tnf-a, Il-6, Il-1b) in liver were evaluated, and the results showed that uoride could deteriorate the in ammation in the HFD mice ( Fig. S4A-F). Furthermore, uoride could upregulate the mRNA expression levels of Myd88 and Tlr4 in liver, suggesting that the exacerbation of obesity by uoride might depend on an activation of TLR4 mechanism.
Fluoride exacerbates the obesity in HFD mice through a TLR4-dependent mechanism As described above, the exacerbation of obesity by uoride might be related to an activation of TLR4 mechanism. Thus, Tlr4 knockout (Tlr4 -/-) mice and wild-type (WT) mice were used to further demonstrate the role of TLR4 in the potential mechanisms (Fig. 3A). As expected, uoride could signi cantly exacerbate the obesity in WT mice (Fig. 3), especially deteriorating the glucose tolerance ( Fig. 3K-L) and inducing lipid accumulation in liver tissues (Fig. 3O). Furthermore, the in ammation, re ecting as increasing levels of TNF-a, IL-6 and IL-1b in plasma, was observed in WT mice ( Fig. S5A-F). However, the exacerbation of obesity and in ammation by uoride was blocked after knockout of Tlr4. The results showed that detrimental effect of uoride on HFD induced obesity was partially dependent on TLR4 mechanism.
Fluoride disturbs the gut microbiota in HFD mice The effect of uoride on the lipid metabolism was investigated using an oleic acid/palmitic acid (OA/PA) induced HepG2 cells or primary murine hepatocytes model (Fig. S6). It was found that uoride showed limited effect on lipid accumulation evaluated by level of TG and oil red O staining. Thus, uoride might not directly lead to exacerbation of the obesity in HFD mice. Recently, evidence is accumulating that dysbiosis of gut microbiota is involved in the development of obesity [7,30]. Moreover, it has been widely reported that uoride could affect the gut microbiota [31,32]. Thus, we suspected that uoride induced changes of gut microbiota might contribute to the exacerbation of the obesity. Fluoride did not exhibit effect on a-diversity of gut microbiota evaluated by observed_otus, Chao1, Shannon and Simpson indexes (data not shown), while b-diversity including principal component analysis (PCA) and Principal coordinates analysis (PCoA) showed that HFD could signi cantly change the structure of gut microbiota ( Fig. 4A and B), and uoride showed remarkable in uence on the composition of gut microbiota (p < 0.01, Fig. S7). At the phylum level, HFD induced a decreased relative abundance of Bacteroidetes and an increased relative abundance of Firmicutes, thereby signi cantly enhanced the ratio of Firmicutes to Bacteroidetes ( Fig. 4C-F), which is in accordance with the previous works [1,9]. Whereas, uoride showed limited effects on the relative abundances of Bacteroidetes and Firmicutes and the ratio of Firmicutes to Bacteroidetes. Then, the linear discriminant analysis effect size (LEfSe) analysis was used to excavate the key gut microbiota changed by uoride ( Fig. 4G-H). It was found that Erysipelotrichaceae at the family level was signi cantly higher than those in the ND and HFD groups. As shown in Fig. 4I, the HFD increased the relative abundance of Erysipelotrichaceae, whereas, uoride could further stimulate the proliferation of Erysipelotrichaceae in the HFD fed mice. Thus, Erysipelotrichaceae at the family level might be the key gut microbiota in the HFD-F group contributing to the exacerbation of obesity.
To further evaluate the relationship between Erysipelotrichaceae, the key candidate gut microbiota, and obesity in human, AGP was applied to demonstrate our prediction [33]. As shown in Fig.5, the relative abundance of Erysipelotrichaceae in overweight and obesity objects was signi cantly higher than that in normal subjects (p < 0.01). Thus, the result further supported our prediction resulting from our analyses in the mice experiments, that Erysipelotrichaceae might be served as the microbial signature of dysbiosis induced by uoride in HFD mice. The Erysipelatoclostridium at the genus level belonging to Erysipelotrichaceae increased signi cantly after uoride exposure compared with that in the ND or HFD group (Fig. 4J). Then, RT-qPCR and species-speci c primers were used to identify the speci c gut microbiota at the species level belonging to Erysipelatoclostridium, it was found that Erysipelatoclostridium ramosum signi cantly increased after exposure of uoride, which might be the potential key bacterium for uoride intervention (Fig. 4K).
Fecal microbiota transplant (FMT) is su cient to induce some phenotypical changes caused by uoride in HFD mice FMT is an effective way to validate the role of the gut microbiota in disease and further identify the speci c bacteria responsible for development of disease [34]. To evaluate whether gut microbiota from uoride-induced mice could lead to obesity, FMT from either the HFD group or HFD-F group mice to antibiotic-treated mice induced by antibiotic cocktail (Abx) was carried out (Fig. 6A). It was found that FMT from uoride-induced mice was su cient to induce some phenotypical changes, mainly including increasing body weight, accumulation of fat tissues, plasma levels of low-density lipoprotein cholesterol (LDL-C), ALT, liver damage and deteriorating the glucose tolerance ( Fig. 6B-O). Although the uorideinduced gut microbiota could not change the colon length, it impaired gut barrier function and promote the in ammation ( Fig. S8). At the same time, the gut microbiota in recipient mice was analyzed (Fig. 7). According to the results of PCA and PCoA, the structure of gut microbiota from recipient mice receiving FMT from HFD or HFD-F group was different ( Fig. 7A-B, p = 0.004 and p = 0.001 for PCA and PCoA, respectively). The relative abundances of Bacteroidetes and Firmicutes and the ratio of Firmicutes to Bacteroidetes showed no signi cant difference between different groups ( Fig. 7C-F). The results of LEfSe analysis showed that Erysipelotrichaceae and Erysipelatoclostridium in recipient mice receiving FMT from the HFD-F group were higher ( Fig. 7G-H), which was further con rmed by statistical analysis (Fig. 7I-J). Thus, the uoride-induced key gut microbiota could colonize in antibiotic-treated mice by FMT, and thereby exacerbated the development of obesity in HFD mice.
The exacerbation of the obesity by uoride is blocked after depletion of the gut microbiota by Abx As described above, the exacerbation of the obesity by uoride might be related to the gut microbiota. Thus, the effect of uoride on the obesity in HFD mice after depletion of the gut microbiota by Abx was performed to investigate whether uoride could exacerbate the obesity in the absence of gut microbiota (Fig. 8A). The level of key gut microbiota was measured by RT-qPCR at the end of experiment, and the results showed that E. ramosum could be scarcely detected (CT value more than 34), which showed that the level of E. ramosum in fecal samples was greatly reduced after depletion of the gut microbiota by Abx. As expected, no remarkably difference was observed in phenotypical changes of obesity after uoride exposure in the absence of gut microbiota (Fig. S9). Furthermore, uoride failed to induce the in ammation after depletion of the gut microbiota (Fig. S10). Thus, the detrimental in uence of uoride on the HFD-induced obesity was blocked after depletion of the gut microbiota, suggesting that the exacerbation of the obesity by uoride was involved in a microbiota-dependent manner.

Fluoride fails to induce the obesity in ND mice
The present result showed that uoride could exacerbate the obesity in HFD fed mice. However, whether uoride could induce the metabolic disease in ND fed mice is still unknown, which was evaluated in the present study (Fig. S10A). Except signi cantly decreasing the plasma level of TC, uoride showed limited effect on the phenotypical changes of obesity ( Fig. S10B-O), indicating that uoride failed to induce the obesity in ND mice. In addition, the gut microbiota was analyzed using 16S rRNA gene sequencing ( E. ramosum aggravates the obesity in HFD mice As described above, Erysipelotrichaceae might be the key bacterium for aggravation of obesity by uoride. Thereinto, E. ramosum, formerly Clostridium ramosum that has been transferred to the new genus Erysipelatoclostridium in the family Erysipelotrichaceae [35], was identi ed as one of the potential key bacteria increased by uoride. Thus, the effect of E. ramosum on the obesity was performed to investigate whether E. ramosum could exacerbate the obesity in HFD mice (Fig. 9A). After intervention with E. ramosum, the body weight, epididymal fat and plasma levels of TC and LDL-C were all signi cantly increased ( Fig. 9B-J). The glucose tolerance and liver brosis were also deteriorated by E. ramosum ( Fig. 9K-O). Furthermore, E. ramosum aggravated the in ammation in HFD mice (Fig. S12). Thus, E. ramosum could aggravate the obesity in HFD mice, which might be regarded as a microbial signature of dysbiosis of gut microbiota induced by uoride.

Fuzhuan brick tea polysaccharides (TPS) abolish the uoride-induced obesity in HFD mice
In recent years, the prebiotics have received increasing attention due to the perfect gut microbiota management with selective promotion of bene cial gut microbiota and inhibition of harmful bacteria [36,37]. Thus, it was expected to explore a novel strategy for prevention of uoride-induced obesity based on the modulation of gut microbiota. In our previous work, TPS could restore the increases in the relative abundance of Erysipelotrichaceae induced by HFD [38]. In the present study, the effect of TPS on uoride induced obesity was investigated (Fig. 9A). Except epididymal and perirenal fat tissues, TPS could signi cantly relieve all the phenotypical changes of obesity induced by uoride ( Fig. 10B-P). Furthermore, TPS could abolish the uoride-induced in ammation in HFD fed mice (Fig. S13). Thus, the intervention of TPS might be served as promising candidate for prevention of uoride-induced obesity.

Discussion
Due to industrial pollution and geological origin, uoride is widespread in our environment, drinking water and various foods [39]. Furthermore, uoride is a widely used additive in mouthwashes and toothpastes to prevent tooth decay and protect our tooth [40]. Thus, the toxicity of uoride has attracted increasing attention in recent years [22]. Thereinto, the uoride-associated hepatotoxicity, impairing glucose tolerance and lipid metabolism disorder have been previously reported [27,41,42], however, the understanding mechanisms of uoride-induced obesity are still not comprehensive, and the role of gut microbiota in the exacerbation of the obesity is ignored. In this work, the potential adverse effect of uoride on the obesity was investigated detailly, furthermore, the potential mechanism was proposed, that uoride exacerbated the obesity in HFD mice via disturbing gut microbiota and driving intestinal barrier permeability.
Firstly, we con rmed that uoride with a dosage of 50 mg/L in the drinking water could signi cantly exacerbate the obesity in mice fed with HFD but not ND according to two separate replicate mice experiments. More speci cally, it was found that uoride further increased body weight, accumulation of fat, liver damage in HFD induced obese mice, whereas no phenotypical change of obesity was observed in ND fed mice after exposure of uoride with same dosage of uoride for 10 weeks. It has been reported that uoride affected the lipid metabolism in a diet-dependent pattern [43]. Fluoride induced steatosis and dyslipidemia in animals fed hypercaloric diets, whereas, no change was observed in normocaloric diet fed rats [28]. Furthermore, uoride with the same dosage could enhance glucose homeostasis along with a normocaloric diet, which might show bene cial effect on diabetes [44]. Likewise, uoride could decrease the level of plasma TC in ND fed mice in this work. Thus, the bene cial or detrimental uoride to health also depended on the diets. In another similar work, foodborne titanium dioxide nanoparticles showed a stronger detrimental effect on colonic in ammation in obese mice than non-obese mice [45]. It should be noted that the uoride concentration of uoride (50 mg/L) used in this work was much higher than that in drinking water between 0.5 and 1.5 mg/L stipulated by World Health Organization (WHO). A lot of reports have demonstrated that moderate level of uoride ingestion not only showed limited detrimental effect on the human health [21,22]. Thus, the effect of low level of uoride ingestion on obesity is still one of the open-ended questions of our study.
The integrity of the epithelial barrier plays an important role in defensing against the invasion of microorganisms and preventing leakage of microbial products such as LPS into the bloodstream [46]. After intestinal barrier dysfunction and dysbiosis, the microorganisms and LPS in bloodstream released from colon could stimulate TLR4, and thereby induce in ammation and metabolic diseases [29,47]. The dietary habits, such as HFD, food additives and probiotics, could affect the intestinal barrier [48]. Recently, some food contaminants, additives or food-derived mycotoxins could directly or indirectly impair the intestinal integrity, and thereby promoting the colitis, liver in ammation or metabolic syndrome [18, 49,50]. The adverse effect of uoride on intestinal barrier has been reported [51,52]. In this work, it was found that uoride could drive the intestinal barrier permeability in mice model, which was further veri ed by in vitro experiment using Caco-2 cells and in vivo experiment using FITC-dextran-based intestinal permeability assay. Moreover, the microorganisms in blood and LPS in plasma were enhanced by uoride exposure. Thus, we hypothesized that microorganisms and LPS in bloodstream could activate TLR4 to cause low-grade in ammation, and thereby exacerbate the obesity. After con rming the upregulation of Tlr4 and Myd88 at mRNA level along with the low-grade in ammation in liver, Tlr4 -/mice and WT mice were applied to demonstrate our hypothesis. Fluoride could still exacerbate the obesity in WT mice, whereas, exacerbation of in ammation and obesity by uoride was blocked in Tlr4 -/mice. Together, these results suggest that uoride exacerbates the obesity in HFD mice via driving the intestinal barrier permeability and activating TLR4 signaling.
In the present work, the limited effect of uoride on the lipid accumulation was observed using HepG2 cells or primary murine hepatocytes model in vitro. Fluoride may not directly affect the lipid metabolism in HFD fed mice. Besides the intestinal barrier permeability as described above, accumulating evidence has demonstrated that the gut microbiota dysbiosis is associated with pathologic conditions of obesity and obesity-related complications [7,30]. Furthermore, the changes of gut microbiota induced by uoride have been reported to be related to its adverse effects, such as neurodevelopmental and cardiovascular impacts [32,53]. Thus, we investigated the potential involvement of the gut microbiota in mediating uoride-induced obesity. Firstly, the results of FMT experiment showed that uoride-induced gut microbiota was su cient to induce some phenotypical changes, suggesting the gut microbiota was involved in uoride-mediated obesity. Furthermore, the exacerbation of obesity by uoride was completely abrogated in mice after depletion of the gut microbiota by Abx, which highlighted that uoride perturbed host-microbiota homeostasis rather than directly triggering host obesity.
Then, 16S rRNA gene sequencing was applied to analyze the changes of gut microbiota after uoride exposure. The results of PCA and PCoA showed that uoride could affect the composition of gut microbiota in HFD fed mice, whereas, it only showed limited effect on the structure of gut microbiota in ND fed mice. Furthermore, it was found that Erysipelotrichaceae at the family level and Erysipelatoclostridium at the genus level were signi cantly increased, which might be responsible for exacerbation of the obesity by uoride exposure. Erysipelotrichaceae could colonize in antibiotic-treated mice by FMT, which might contribute to the obesity in recipient mice. The potential role of Erysipelotrichaceae in in ammation and metabolic disorders have been widely reported [54,55], which was summarized by Kaakoush [56]. Importantly, the HFD could increase the abundance of Erysipelotrichaceae [38], then uoride could further promote the proliferation of Erysipelotrichaceae, thereby exacerbated the HFD-induced obesity. Interestingly, uoride failed to promote the proliferation of Erysipelotrichaceae and Erysipelatoclostridium in the absence of HFD, which might explain why uoride could not induce obesity in ND mice. Thus, the changes of composition and structure in gut microbiota and the proliferation of Erysipelotrichaceae and Erysipelatoclostridium by uoride were diet-dependent. In a similar work, uoride also showed limited effect on gut microbial communities in mice fed on standard mouse chow [57].
Based on the data from database of AGP, the relative abundance of Erysipelotrichaceae in overweight and obese people was signi cantly higher than those in people with normal BMI, further veri ed the key role of Erysipelotrichaceae in development of obesity. E. ramosum belonged to Erysipelotrichaceae was identi ed as one of potential key bacteria contributing to detrimental effect of uoride on obesity. E. ramosum was associated with systemic low-grade in ammation, metabolic parameters and corpulence traits [58,59]. Furthermore, the promotion of HFD-induced obesity in mice has been reported after administration of E. ramosum [60,61]. In this work, E. ramosum could exacerbate the in ammation and obesity in HFD mice. Moreover, E. ramosum showed a positively association with many diseases, such as COVID-19 disease and colorectal cancer [62, 63], which has been regarded as a microbial signature of microbiota dysbiosis. Thus, we would like to emphasize that E. ramosum was one of key gut microbiota contributing to detrimental effect of uoride on obesity, which could be considered as a microbial signature of uoride-induced microbiota dysbiosis. Unfortunately, the relative abundance of Erysipelotrichaceae in human feces from database of AGP is not informative on relative abundance of E. ramosum. Thus, whether other bacteria belonged to Erysipelotrichaceae being responsible for exacerbation of the obesity by uoride is still one of the open-ended questions of our study.
Prebiotics, one microbiota-management tool, could inhibit the harmful pathogen, and stimulate the proliferations of bene cial bacteria, thereby improve human health [36,37]. Thus, the development of microbiota-directed dietary bers is expected as a novel strategy to prevent the gut microbiota-related diseases [64, 65]. As described above, the exacerbation of the obesity in HFD mice might depend on the bloom of Erysipelotrichaceae after exposure of uoride. Thus, it was expected to explore a novel dietary ber to inhibit the proliferation of Erysipelotrichaceae, thereby prevent the uoride-induced exacerbation of obesity. In our previous work, TPS treatment could prevent the proliferation of Erysipelotrichaceae in HFD induced obese mice [38]. As expected, TPS could abolish the uoride-induced obesity in HFD mice in this work. Some functional foods also showed the ability to reduce the uoride-induced detrimental effects. For example, rutin could protect uoride induced dyslipidemia, blood toxicity and cardiotoxicity in rats [66]. Thus, we open up exciting therapeutic avenues for prevention of uoride-induced obesity, which used TPS as a potential candidate for modulation of gut microbiota.
In conclusion, it was found in the present study that uoride, widely consumed by humans, could exacerbate the obesity in HFD fed mice, while the uoride alone did not show detrimental effect on the obesity in ND fed mice. Furthermore, we provided the rst evidence of a comprehensive cross-talk mechanism between uoride and obesity in HFD fed mice, which was mediated by gut microbiota and intestinal barrier. Another nding is that Erysipelatoclostridium ramosum was identi ed as a crucial mediator of uoride induced obesity, which could be explored as potential target for prevention and treatment of obesity with exciting translational value. Finally, TPS is suggested as a potential candidate to prevent uoride induced obesity.  Supplementary Table 1. Before experiment, all animal underwent a 1-week acclimatization period fed with ND. The uoride was administrated to mice by addition to drinking water, and the drinking water was refreshed twice one week. The food intake and body weight were recorded once a week during the animal experiments.

Methods
Animal protocol 1. The purpose of this rst mice experiment was to evaluate the effect of uoride on the obesity in HFD mice. 24 male C57BL/6J mice with 6 weeks of age were randomly divided into three groups including the ND, HFD, and HFD-F groups (HFD plus 50 mg/L of uoride in drinking water) (n = 8 per group). The intervention continued for 10 weeks, and the mice were euthanized after fasting overnight at the end of the experiment.
Animal protocol 2. The purpose of this mice experiment was to con rm the effect of uoride on the obesity in HFD mice in a separate replicate experiment. Furthermore, the potential mechanisms were investigated based on the rst mice experiment, which focused on the gut microbiota and intestinal barrier permeability. 24 male C57BL/6J mice with 5 weeks of age were randomly divided into three groups including ND, HFD, and HFD-F groups (n = 8 per group). The intervention continued for 10 weeks. The fecal samples were collected for 16S rRNA sequencing at the last week, and the mice were Animal protocol 4. The purpose of this mice experiment was to investigate whether the knockout of Tlr4 could block the exacerbation of obesity by uoride in HFD mice. Sixteen WT and sixteen Tlr4 -/mice (C57BL/10J, 5 weeks old) were randomly divided into two groups, respectively, including HFD and HFD-F (HFD plus 50 mg/L of uoride in drinking water) groups (n = 8 per group). The intervention continued for 8 weeks, and the mice were euthanized after fasting overnight at the end of the experiment.
Animal protocol 5. The purpose of this mice experiment was to investigate whether uoride-induced gut microbiota could be su cient to induce some phenotypical changes of obesity by FMT. The mice fed HFD or HFD plus 50 mg/L of uoride in drinking water described in Animal protocol 2 was chosen as donor. Another 16 male C57BL/6J mice with 5 weeks of age were randomly divided into two groups (n = 8). At rst two weeks, all mice were treated by Abx (containing 10 g/L of vancomycin hydrochloride, 20 g/L of neomycin sulfate, 20 g/L of metronidazole and 20 g/L of ampicillin sodium salt) by gavage with dosage of 200 mL for each mouse once daily according to the previous work to obtain the antibiotictreated mice [49,68]. At third week, FMT was carried out by transplanting the fecal slurry from donor mice to the antibiotic-treated mice (n = 8) until the end of the experiment as described previously [2,69]. Brie y, the fecal samples collected from donor mice (HFD and HFD-F groups) were immediately pooled and diluted in sterile saline containing 0.5 g/L cysteine hydrochloride at proportion of 100 mg/mL. Then, the fecal slurry was centrifuged at 1000 r/min for 1 min to obtain fecal supernatant. Each mouse received 200 mL of the fecal supernatant from HFD or HFD-F groups by gavage, respectively. The FMT continued for 8 weeks, and the mice were euthanized after fasting overnight at the end of the experiment.
Animal protocol 6. The purpose of this mice experiment was to investigate whether uoride could exacerbate the obesity in the absence of gut microbiota. Sixteen male C57BL/6J mice with 5 weeks of age were randomly divided into two groups including HFD fed with Abx (HFD-Abx) group, and HFD fed with Abx plus 50 mg/L of uoride in drinking water (HFD-Abx-F) group (n = 8 per group). Abx was treated to mice by gavage once daily to clear gut microbiota according to the previous work with some modi cations [68,70]. The intervention continued for 10 weeks, and the mice were euthanized after fasting overnight at the end of the experiment. Animal protocol 7. The purpose of this mice experiment was to investigate whether uoride could induce the obesity in ND mice. Sixteen male C57BL/6J mice with 5 weeks of age were randomly divided into two groups including ND group, and ND plus 50 mg/L of uoride in drinking water (ND-F) group (n = 8 per group). The intervention continued for 10 weeks, and the mice were euthanized after fasting overnight at the end of the experiment.
Animal protocol 8. The purpose of this mice experiment was to investigate whether E. ramosum could exacerbate the obesity in HFD mice (result obtained in animal experiment 2 and 5). Antibiotic-treated mice were obtained by treatment of Abx for rst two weeks as described in Animal protocol 5. Then, antibiotic-treated mice were randomly divided into two groups including HFD fed with PBS and HFD fed with E. ramosum (HFD-ER) by intragastric gavage for two weeks. Each mouse received 0.2 mL of live E. ramosum (4 × 10 8 cfu/mL) daily. The intervention of E. ramosum continued for 2 weeks. After further 4 weeks, the mice were euthanized after fasting overnight at the end of the experiment.
Animal protocol 9. The purpose of last mice experiment was to present a potential strategy to prevent uoride-induced exacerbation of obesity in HFD mice by TPS. Sixteen male C57BL/6J mice with 5 weeks of age were randomly divided into two groups including HFD plus 50 mg/L of uoride in drinking water (HFD-F) group, and HFD plus 50 mg/L of uoride in drinking water and 400 mg/kg/day of TPS (HFD-F-TPS) group by intragastric gavage (n = 8 per group). The mice in HFD-F group received same volume of water by intragastric gavage. The intervention continued for 8 weeks, and the mice were euthanized after fasting overnight at the end of the experiment. The lipid metabolism assay in HepG2 cells or primary murine hepatocytes model in vitro HepG2 cells were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). HepG2 cells were incubated in DMEM supplemented with 10% of fetal bovine serum, and 1% of penicillinstreptomycin, at 37 ℃under 5% of CO 2 atmosphere. The cytotoxity of uoride on HepG2 cells was investigated by MTT assay as described for Caco-2 cell. The effect of uoride on the lipid metabolism was evaluated in HepG2 cells in vitro as described previously with some modi cations [73]. HepG2 cell suspension was plated in a 12-well plate with 1 mL/well, and cultured for 24 h. Then, the serum-free medium, containing 100 mM of palmitic acid, 200 mM of oleic acid and different dosages of uoride (0, 10 and 50 mg/L, respectively), was added to replace the old medium, the cells were further incubated for 24 h to induce excess fat synthesis. After washed by PBS for three times, the cells were stained with Oil Red O solution to evaluate the level of intracellular fat droplets. Furthermore, the Oil Red O was extracted by 100% isopropyl alcohol, and the absorbance of the extracted isopropyl alcohol solution was measured at 490 nm to further quantify the intracellular lipid content. Moreover, the triglyceride (TG) and protein contents in HepG2 cells were measured by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The primary murine hepatocytes were isolated from the liver of C57BL/6 mice using a collagenase perfusion method according to the reported work [74]. After isolation from mice, the cell suspension was inoculated in a 12-well plate with 1.0 mL/well. After cultured for 24 h, the effect of uoride on the lipid metabolism in primary murine hepatocytes was investigated as mentioned above for HepG2 cells model.

Cultivation of Erysipelatoclostridium ramosum
E. ramosum CCUG35705 was purchased from Culture Collection University of Gothenburg (CCUG, Sweden). E. ramosum was cultured in brain heart infusion (BHI) medium at 37 ℃ at an anaerobic workstation (HYQX-, Shanghai Yuejin Medical Instrument Co., Ltd., Shanghai, China). After incubation for 12 h, the E. ramosum was centrifuged at 4000 g for 5 min, and resuspended in anaerobic PBS containing 4 × 10 8 cfu/mL of E. ramosum. Then, the E. ramosum would be gavaged to mice immediately.

Oral glucose tolerance test (OGTT)
One week before the end of animal experiment, the mice were fasted overnight for OGTT. After measurement of blood glucose, the mice were immediately given an oral gavage glucose at a dosage of 1.5 g/kg body weight. Then, the blood glucose was further measured after 15, 30, 60, 90 and 120 min. All blood samples were collected from the tip of the tail vein, and the blood glucose was measured using a glucose meter (Sinocare Inc., Changsha, Hunan, China).

Tissue sampling
The animals were anesthetized with carbon dioxide, and blood, liver, colon, perirenal fat, mesentery fat, and epididymal fat tissues were collected. The blood sample was collected in sterile anticoagulation tube (BD Biosciences, USA). Then, 50 mL of blood was immediately taken out and stored at -80 ℃ for quanti cation of Bacterial DNA. The residue blood was centrifuged at 3000 r/min for 15 min at 4 ℃ to afford plasma. After weighing and taking picture, a part of the colon and epididymal fat tissues were xed in 4% paraformaldehyde solution for histological analysis. Other tissues were stored at -80 ℃ for further experiment.

Biochemical analyses
The contents of TG, TC and LDL-C in plasma and TG in liver were measured by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The levels of TNF-a, IL-6 and IL-1b in plasma were determined using commercial ELISA kits (Neobioscience Technology Co, Ltd., Shenzhen, China). Plasma LPS was determined using commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The insulin in plasma was determined using commercial ELISA kit (Mercodia AB, Uppsala, Sweden).

Quanti cation of bacterial DNA in blood
The quanti cation of bacterial DNA in blood were carried out according to previous work with some modi cations [67]. The whole DNA in blood (50 mL) was extracted using a commercially available kit  Table 2.
Histology analysis and immuno uorescence assessment The histological analyses of epididymal fat and colon were carried out according to the previous work [14,38]. After xed in 4% paraformaldehyde solution, the epididymal fat and colon tissues were embedded in para n, and stained with hematoxylin and eosin (H&E). The protein expressions of ZO-1, Occludin, Claudin-1 and MUC1 in the colon tissue were also evaluated by immuno uorescence assessment as previously described [74]. After depara nized and rehydrated, the colon samples were incubated twice in xylene for 15 min each, dehydrated twice in pure ethanol for 5 min each, dehydrated in gradient ethanol of 85% and 75% ethanol for 5 min each, and washed in distilled water, respectively. The samples were dipped in 3% of bull serum albumin (BSA) for 30 min to block non-speci c binding. The BSA solution was discarded, the primary antibody, including Anti-ZO-1 (ab221547), Anti-Occludin

Human cohort analyses
The data from the American Gut Project (AGP) was reanalyzed to gain insight into the relationship between the relative abundance of Erysipelotrichaceae and obesity according to the previous work [76,77]. The subjects which contained fecal samples were chosen. The subjects which contained fecal samples were chosen. Subjects who were treated with antibiotic or had diabetes were excluded, and the samples with missing information such as BMI and sex were removed. Furthermore, the subjects whose BMI were more than 50 or less than 18.5 kg/m 2 were also excluded. Finally, a total of 10,376 individuals aged from 20 to 99 years, including 6546 normal, 2869 overweight and 961 obesity subjects, were obtained in this work. The sequence read les of these subjects were obtained from European Bioinformatics Institute (EBI, PRJEB11419) [33]. The sequences were analyzed using pipeline as previously described       . the relative abundances of (I) Erysipelotrichaceae, and (J) Erysipelatoclostridium, (K) Level of E. ramosum in fecal samples measured by RT-qPCR using species-speci c primers. Higher CT values suggest lower levels of E. ramosum. The results were expressed as means ± SEM. Statistical signi cance was carried out by one-way ANOVA with Tukey test. Adjusted p-values (q-values) were used to evaluate differences in analysis of gut microbiota based on false discovery rate (FDR) for multiple testing according to the Benjamini and Hochberg procedure. A value of p or q < 0.05 was considered to be signi cant.

Figure 5
The statistical analysis of Erysipelotrichaceae among subjects with different BMI categories based on the database of American Gut Project (AGP  expressed as means ± SEM. Difference in two groups was calculated using the Mann-Whitney test or Kruskal-Wallis test. A value of p < 0.05 was considered to be signi cant.

Figure 7
The uoride-induced key gut microbiota could colonize in antibiotic-treated mice by FMT.    Plasma ALT. The results were expressed as means ± SEM. Difference in two groups was calculated using the Mann-Whitney test or Kruskal-Wallis test. A value of p < 0.05 was considered to be signi cant.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementaryinformationforMicrobiomeXZeng.docx