Microbiota depletion of conventional mice raises plasma cholesterol level

We aimed to decipher the role played by the intestinal microbiota in the regulation of plasma cholesterol levels in mice. To address this question, we depleted the gut microbiota of spontaneously hypercholesterolemic Apoe−/− mice over 4 weeks by daily gavage with a mixture of antibiotics consisting of vancomycin, ampicillin, neomycin, and metronidazole (Fig. 1a, Additional file 1). After 7 days of treatment, intestinal microbiota depletion was effective and stable during 3 weeks with a copy number of 16S rRNA genes in feces 105-fold less than the initial bacterial load (Additional file 2: Figure S1) in agreement with previous findings [31]. Plasma total cholesterol level was 55% higher in microbiota-depleted (AB-Mdpl) mice compared with conventionally raised (Conv-R) mice (Fig. 1b). Plasma phospholipids and triglycerides were also raised by microbiota depletion, although not statistically significant for triglycerides (Fig. 1b).

Fig. 1 Intestinal microbiota depletion raises plasma cholesterol levels and intestinal cholesterol absorption. a Experimental design. See also Additional file 2: Figure S1. b Plasma cholesterol, phospholipids and triglycerides levels in conventionally raised (Conv-R) and microbiota-depleted mice (AB-Mdpl). c Cholesterol distribution across the VLDL, LDL, and HDL lipoprotein classes analyzed by fast protein liquid chromatography. d Plasma radioactivity 2 h after gavage with [3H]-cholesterol. e Relative expression of genes related to cholesterol absorption in the jejunum. f Relative expression of genes related to lipoprotein secretion in the jejunum. Data are represented as mean ± SEM, n = 5–10 mice/group (d, e) or as dots with median (b–f). Data were analyzed with Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

Cholesterol in the plasma exists mainly packaged in the form of lipoproteins: chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Quantitative analysis of cholesterol distribution among lipoproteins separated by gel filtration revealed an increase of the abundance of VLDL and LDL subclasses (respectively + 53% and + 36%) in AB-Mdpl mice, whereas the HDL fraction was similar in Conv-R and AB-Mdpl mice (Fig. 1c).

These experiments confirm that intestinal microbiota contribute to the regulation of plasma cholesterol levels and demonstrate that microbial depletion strongly affect several lipoproteins levels, mainly VLDL and LDL.

Intestinal microbiota depletion increases intestinal cholesterol absorption with no effect on hepatic VLDL production

As the liver secretes VLDL particles, we investigated the impact of microbiota depletion on VLDL production. Likewise, as LDL particles derive from the loss of triglycerides by VLDL and intestine originating chylomicrons in the bloodstream, we investigated intestinal cholesterol absorption.

Then, we measured the appearance of labeled cholesterol in the plasma of conventionally raised and microbiota-depleted mice 2 h following gavage of [3H]-cholesterol in olive oil. The appearance of radiolabeled cholesterol in the plasma was 25% higher in antibiotic-treated mice (Fig. 1d), indicating that depleting the microbiota raises intestinal cholesterol absorption.

We next analyzed the jejunal expression of genes involved in intestinal cholesterol absorption (Npc1l1 [39]) and intracellular cholesterol excretion in the gut lumen (Abcg5 and 8 [40]). We observed that microbiota-depleted mice displayed a threefold increase in Npc1l1 expression while Abcg8 expression was moderately raised and Abcg5 expression was not affected (Fig. 1e). Moreover, the expression of several genes encoding apolipoproteins and proteins involved in chylomicron and preβ-HDL assembly and secretion were increased at least two folds in the jejunum of microbiota-depleted mice (Fig. 1f).

VLDL are assembled in the liver from triglycerides, cholesterol, and apolipoproteins (ApoB mainly) by the chaperone Mttp. Here, liver gene expression levels of ApoB and Mttp of Conv-R and AB-Mdpl mice were similar (Additional file 3: Figure S2A). This is consistent with the similar VLDL secretion rate assessed using Triton WR-1339 as an inhibitor of peripheric lipid uptake by endothelial lipoprotein lipase [41] (Additional file 3: Figure S2B).

This set of experiments reveals that depleting the intestinal microbiota with antibiotics raises intestinal cholesterol absorption. On the contrary, the hypothesis of elevated VLDL levels in microbiota-depleted mice being a consequence of increased hepatic VLDL synthesis and secretion is rather unlikely.

Intestinal microbiota depletion increases the hepatic clearance of plasma cholesterol through LDLr

[3H]-cholesterol absorption assay demonstrated that the level of radiolabeled cholesterol was 37% higher in the liver of microbiota-depleted mice (Fig. 2a, Additional file 4), suggesting a microbial regulation of hepatic cholesterol uptake. The uptake of cholesterol-rich particles HDL and LDL into the liver is mediated by their respective receptors, scavenger receptor type B1 (SR-B1) and LDL receptor (LDLr) [42]. mRNA levels of LDLr were significantly increased by microbiota depletion which was not the case for SR-B1 mRNA (Fig. 2b). Hence, we submitted LDLr−/− mice to the same microbiota depletion protocol and measured their circulating cholesterol levels. Strikingly, microbiota depletion raised plasma cholesterol levels by 91% in LDLr-deficient mice against only 50% in Apoe-deficient mice (Fig. 2c). This demonstrates that LDLr-mediated cholesterol uptake by the liver partially counteracts the plasma cholesterol raise induced by microbiota depletion.

Fig. 2 Intestinal microbiota depletion increases hepatic cholesterol uptake and hepatic cholesterol synthesis. a Liver radioactivity 2 h after gavage with [3H]-cholesterol in conventionally raised (Conv-R) and microbiota-depleted mice (AB-Mdpl). b Hepatic relative expression of cholesterol transporters. c Plasma cholesterol increase in microbiota-depleted mice in comparison to control mice in Apoe (○) and LDLr (□)−/− mice. d Hepatic relative expression of genes related to cholesterol synthesis. See also Additional file 5: Figure S3. e Cholesterol and lathosterol concentration analyzed by GC-MS in the liver. Data are represented as mean ± SEM, n = 6–9 mice/group (b–d) or as dots with median (a, c, e). Data were analyzed with Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

Intestinal microbiota depletion enhances cholesterol synthesis in the liver

The gastrointestinal tract contributes to 15–35% and the liver to 20–40% of total cholesterol synthesis in rodents [43]. The relative expression of Hmgcs1 and HmgcoAr, encoding two key enzymes in cholesterol biosynthesis pathway, was not affected following intestinal microbiota depletion in the intestine (Additional file 5: Figure S3) but significantly increased by four- to sevenfold in the liver (Fig. 2d). We next determined the liver content of cholesterol and lathosterol, a synthesis intermediate considered as a marker of cholesterol synthesis [44], by gas chromatography coupled to mass spectrometry (GC-MS). Cholesterol concentration was 30% higher and lathosterol concentration was doubled in the liver of AB-Mdpl compared to Conv-R mice (Fig. 2e). This indicates that intestinal microbiota regulates cholesterol biosynthesis specifically in the liver.

The intestinal microbiota influences bile acid synthesis and biliary cholesterol secretion

Cholesterol is mainly excreted from the body in the bile that is then secreted in the duodenum, leading to fecal excretion in two forms: cholesterol and bile acids. To evaluate cholesterol output from the liver, we monitored bile flow during 1 h and found a 40% increase in AB-Mdpl mice compared to control mice (Fig. 3a, Additional file 6). We demonstrated that biliary cholesterol secretion in the intestinal lumen was significantly increased in AB-Mdpl mice compared to controls (Fig. 3b). Importantly, cholesterol is apically secreted from hepatocytes to bile as free cholesterol via ABCG5/8 [45], whose gene expression was twofold greater in AB-Mdpl mice (Fig. 3c).

Fig. 3 Enterohepatic cycle of cholesterol and bile acids in conventionally raised and microbiota-depleted mice. a Bile volume collected in 1 h of gallbladder cannulation in conventionally raised (Conv-R) and microbiota-depleted mice (AB-Mdpl). b Quantity of cholesterol secreted in the bile during 1 h of gallbladder cannulation. c Hepatic gene expression of enzymes involved in bile acid biosynthesis and of transporters of cholesterol and bile acids in conventionally raised (Conv-R) and microbiota-depleted mice (AB-Mdpl). d Fecal excretion of 14C bile acids (water-soluble fraction) and 14C cholesterol (cyclohexane soluble fraction) during 72 h after oral gavage with 14C cholesterol. e 14C bile acids excreted in the feces expressed as percent of total radioactivity (cholesterol + bile acids). f Relative expression of fgf15 in the distal ileum. g Plasma radioactivity 2 h after gavage with [3H]-taurocholic acid. h Relative gene expression of bile acid transporters in the distal ileum. Data are represented as mean ± SEM (c, f, h) or as dots with median (a, b, g), n = 5–8 mice/group. Data were analyzed with Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

The drastic depletion of intestinal microbiota increases intraluminal cholesterol absorption as well as re-excretion in the bile by the liver. To investigate how intestinal microbiota depletion influences the balance between cholesterol intake and secretion, we force-fed mice with 14C-cholesterol and collected their feces every 24 h during 72 h. We separated neutral lipids containing cholesterol from water-soluble components including bile acids and measured radioactivity in each fraction (Fig. 3d). Conv-R mice excreted 70% more radioactive sterols (sum of neutral lipids and water-soluble fraction) than AB-Mdpl over 72 h (Fig. 3d), confirming that sterols accumulated in the body in the absence of microbiota. Specifically, AB-Mdpl mice excreted threefold less cholesterol and 50% more bile acids than Conv-R mice; hence, the bile acids represented a significantly higher proportion of fecal sterols in Ab-Mdpl mice (Fig. 3e). This suggests that the absence of gut microbiota leads to an accumulation of sterols in the body and that in this context bile acids constitute a significant proportion of fecal sterols.

Next, we observed that the increased fecal bile acid excretion was associated with a regulation of enzymes in the bile synthesis pathway. Expression levels of Ak1r1d1 and Cyp7a1, the rate-limiting enzyme in the bile acid synthesis pathway, were increased in the liver in AB-Mdpl mice, supporting an increased bile acid synthesis in the absence of microbiota (Fig. 3c). However, Cyp27a1 expression was similar in both groups while Cyp8b1 expression was decreased in AB-Mdpl mice (Fig. 3c). Considering that microbiota is known to induce intestinal FXR which in turn regulates hepatic Cyp7a1 through a fibroblast growth factor 15 (Fgf-15)-dependent mechanism [46], we determined Fgf-15 expression in the distal ileum. We found that microbiota depletion reduces Fgf-15 expression by 75% (Fig. 3f).

As microbiota depletion raises bile acid synthesis and secretion, we needed to examine whether modification of intestinal bile acid absorption can strengthen or lessen fecal loss of bile acids. Gavage with 3H-taurocholic acid showed that microbiota depletion significantly decreases taurocholic acid absorption (Fig. 3g). This is probably not related to a decrease in active transport of bile acids, as the gene expression of the two transporters Ibat and Abcc3 was not decreased by the microbiota depletion (Fig. 3h). This decrease in taurocholic acid absorption is therefore likely the consequence of a decrease in passive absorption, the major absorption pathway of microbiota-derived unconjugated bile acids [47].

Plasma cholesterol level is transmissible from humans to mice by microbiota transplantation

Our first experiments indicated that the lack of a functional microbiota deeply disrupts host cholesterol metabolism. We therefore hypothesized that not only bacterial load will impact cholesterol metabolism, but also that variations in intestinal microbiota composition and functionality might induce variations of cholesterol circulating levels. We thus selected human microbiota donors whose plasma cholesterol levels were discrepant and colonized recipient mice with their intestinal microbiota. We selected four women based on their plasma lipid profile: two donors with normal blood cholesterol levels (NorChol) and two donors with moderately elevated total cholesterol levels (HiChol) (Fig. 4a, Additional file 7). These subjects received no treatment. Consistently with a dyslipidemic context, HDL cholesterol levels were slightly lower in the two HiChol donors while LDL cholesterol and triglycerides levels were considerably higher (Fig. 4a).

Fig. 4 Plasma cholesterol levels are transferable from humans to mice by intestinal microbiota transplantation. a Donors’ characteristics and experimental design. b Plasma cholesterol, phospholipids, and triglycerides levels in mice colonized with the microbiota from normocholesterolemic donors (NorChol-r1 and r2, pictured cyan and dark cyan) and high-cholesterol donors (HiChol-r1 and r2, pictured in red and dark red). Data are represented as dots with median (a, b), n = 8–12 mice/group. Recipient groups were analyzed using Kruskal–Wallis test followed by Dunn’s pairwise multiple comparison procedure. *q < 0.05, **q < 0.01, ***q < 0.001 Full size image

We colonized four groups of microbiota-depleted 7-week-old female Apoe−/− mice (n = 10–14 mice per group) through repeated oral gavages with fecal microbiota from respective donors (Fig. 4a). Strikingly, after 9 weeks, the mean of plasma total cholesterol levels of HiChol recipient mice was 23% higher than those of NorChol recipients (Fig. 4b). Other plasma lipids such as triglycerides and phospholipids were also dramatically increased (Fig. 4c), suggesting that as their donors, HiChol recipient mice had an overall altered plasma lipid profile.

Intestinal microbiota regulates cholesterol absorption/synthesis balance

To investigate if intestinal microbiota from dyslipidemic or normolipidemic donors could modulate cholesterol metabolism pathways, we analyzed the expression in the jejunum of genes related to intestinal cholesterol absorption and lipoprotein secretion. Npc1l1, ApoB, ApoCII, and Mtpp were all significantly more expressed in both HiChol recipient groups than in both NorChol recipient groups (Fig. 5a, Additional file 8). This suggests that the intestinal microbiota from dyslipidemic donors upregulates intestinal cholesterol absorption in recipient mice compared to mice colonized with microbiota from normolipidemic donors.

Fig. 5 Intestinal microbiota regulates cholesterol absorption/synthesis balance. a Relative expression of genes related to cholesterol absorption and lipoprotein secretion in the jejunum in mice colonized with the microbiota from normocholesterolemic donors (NorChol-r1 and r2, pictured cyan and dark cyan) and high-cholesterol donors (HiChol-r1 and r2, pictured in red and dark red). b Relative expression of enzymes involved in cholesterol synthesis in the liver. See also Additional file 9: Figure S4. c Cholesterol and lathosterol concentration analyzed by GC-MS in the liver. d Triglycerides and phospholipids analyzed by biochemic assay in the liver. e Hepatic relative expression of LDLr. f Hepatic relative expression of Cyp7a1 in the liver. g Relative expression of fgf15 in the distal ileum. Data are represented as mean ± SEM (a, b, e, f, g) or as dots with median (c, d), n = 8–12 mice/group. Recipient groups were analyzed using Kruskal–Wallis test followed by Dunn’s pairwise multiple comparison procedure. *q < 0.05, **q < 0.01, ***q < 0.001 Full size image

On the contrary, genes of the cholesterol synthesis pathway (HmgcoAr and Hmgcs1) were two times less expressed in the liver of HiChol recipients than in Norchol recipients (Fig. 5b). Consistently, the concentration of lathosterol was significantly lower in the liver of the two groups of HiChol recipients than in the liver of Norchol recipients, supporting a decrease in hepatic cholesterol synthesis in HiChol recipient mice (Fig. 5c). However, hepatic cholesterol content was not affected by the donors’ status (Fig. 5c), suggesting that other cholesterol metabolism pathways in the liver were affected by the microbiota. As cholesterol, liver phospholipids were similar in the four groups while liver triglycerides were slightly raised in HiChol recipients in comparison to NorChol recipients (Fig. 5d).

Hepatic expression of LDL receptor was lower in HiChol than in NorChol recipient mice (Fig. 5e), suggesting a decreased hepatic uptake in mice colonized with the microbiota from dyslipidemic donors. Moreover, the expression of Cyp7a1 was also reduced in HiChol recipients, which likely result from the increased expression of its suppressor Fgf15 in the distal ileum (Fig. 5f, g). There was a trend towards decreased Cyp8b1 and canalicular cholesterol Abcg5/g8 and bile acid Abcb11 transporters, but this did not reach statistical significance (Additional file 9: Figure S4).

Altogether, this set of experiments suggests an elevated intestinal cholesterol absorption and a decreased hepatic uptake and synthesis in HiChol recipient mice in comparison to NorChol recipient mice. Biliary cholesterol secretion in the intestinal lumen may also be lower in HiChol than in NorChol recipient mice. This indicates more broadly that the microbiota could be a regulator of the intestinal absorption/hepatic synthesis balance.

Mice colonized with the microbiota of normocholesterolemic and dyslipidemic human donors harbor distinct intestinal microbiota composition

In order to identify bacterial species or taxa involved in the regulation of cholesterol homeostasis, we analyzed by 16S rRNA gene sequencing of the V3-V4 region the fecal microbiota of recipient mice 9 weeks after colonization. Richness, Simpson, and Shannon alpha diversity indices were similar between recipient mice groups (Additional file 10: Figure S5). Interclass PCA based on the ASV abundance showed that the microbiota of mice clustered separately depending on the microbiota donor (Fig. 6a). The two NorChol and the two HiChol recipient groups did not cluster together. We then looked for ASVs that were specifically over- or underrepresented in both NorChol groups in comparison to both HiChol groups, and no particular phylum was differently represented in NorChol and HiChol recipient mice (Fig. 6b and Additional file 11: Figure S6). After assignation to lower taxonomic levels and cladogram construction using GraPhlAn [38], we found that Betaproteobacteria class was significantly more abundant in both HiChol recipient groups of mice than in both NorChol recipient mice groups (Fig. 6b and Additional file 11: Figure S6). This was mainly due to higher proportions of unclassified Betaproteobacteria (Fig. 6b and Additional file 12: Figure S7). Unclassified Firmicutes were also found in higher proportions in the microbiota of HiChol recipient mice (Fig. 6a, c, d, and Additional file 12: Figure S7 A and B). Ten ASVs corresponding to 6 taxonomic clusters were found to be more abundant in HiChol recipient’s microbiota (Fig. 6c). Three members of the Bacteroidales S24-7 class were more abundant in HiChol recipients than in NorChol recipients, as well as one ASV related to Bacteroides genus, one related to Alistipes genus and Barnesiella genus (Fig. 6c). In addition, 3 ASVs belonging to unclassified Betaproteobacteria and one to unclassified Firmicutes were specifically associated with HiChol recipients.

Fig. 6 Mice colonized by the microbiota of normocholesterolemic and high-cholesterol human donors harbor specific intestinal microbiota composition. a Interclass principal component analysis performed based on ASVsabundance. Mice microbiota were clustered and the center of gravity computed for each group. The p value of the link between recipient groups and ASV abundance was calculated using a Monte Carlo test (999 replicates). b Cladogram generated using GraPhlAn [38] representing recipients’ microbiota with cyan clade-markers highlighting bacterial groups significantly more abundant in NorChol recipients and red clade-markers highlighting bacterial groups significantly more abundant in HiChol recipients as assessed by Kruskal–Wallis test followed by Dunn’s pairwise multiple comparison procedure. Circular heatmap represents normalized abundance of all ASV in each recipient group, with the darkest color corresponding to the group having the highest percentage of the given ASV. Black bars represent the mean abundance of the ASVs in the whole data set. c Bacterial ASVs statistically more abundant in both HiChol recipients’ groups than in both NorChol recipients’ groups. n = 9–12 mice/group. d Spearman correlations between ASV-level microbial populations and cholesterol metabolism-associated parameters. Strong correlations are indicated by large circles, whereas weaker correlations are indicated by small circles. The colors of the circles denote the nature of the correlation with dark blue indicating strong positive correlation and dark red indicating a strong negative correlation. ¤q < 0.05, ¤¤q < 0.01, ¤¤¤q < 0.001 after FDR correction Full size image

HiChol-associated microbiota taxa correlate with plasma cholesterol levels

To confirm whether one or several specific gut bacteria were involved in the regulation of major cholesterol metabolism pathways, we performed multiple correlation analyses between the previously identified ASVs and plasma cholesterol level as well as parameters associated with hepatic cholesterol synthesis, lipoprotein uptake by the liver, bile acid synthesis, and intestinal absorption (Fig. 6d). Six of the ten HiChol recipient-associated ASVs were significantly and positively correlated with plasma cholesterol levels. Five of these ASVs correlated negatively with markers of hepatic cholesterol synthesis such as HmgcoAr expression and lathosterol concentration in the liver. These ASVs also positively correlated with markers of intestinal absorption such as Npcl1 and Mttp expression in the jejunum. The Fgf15 expression in the ileum and the LDLr expression in the liver were also correlated with these ASVs; however, statistical significance was not reached, suggesting that these parameters of cholesterol metabolism are less tightly regulated by the microbiota than the other parameters. The sequences of seven of these ten ASVs were not assigned to the genus level by Qiime2 pipeline; however, manual BLAST against the EzBioCloud 16S data base (update 06 august 2019) [48] indicated that ASV 1 belongs to the Sutterellaceae family, ASV 3 and ASV 8 belong to the Turicimonas genus, and ASV 4 to the Erysipelotrichaceae family.