Animals and diets

All the mice used in this study were wild-type (WT) on a C57BL/6 background and bred at the Massachusetts General Hospital (MGH) animal facility or purchased from Charles River Laboratories. Mice were housed in a biosafety level 2 room in hard top cages with two or three mice per cage. Mice were maintained in a temperature-controlled room (22–24 °C) with a 12-h light/12-h dark diurnal cycle and allowed for food and water ad libitum. Diets used in this study were either normal chow diet (CD) (Laboratory Rodent Diet 5001) from LabDiet or western diet (WD) (D12079B) from Research diets, Inc., NJ, USA. All animal procedures in this study were carried out in accordance with the guidelines approved by the MGH Subcommittee on Research Animal Care.

Animal experiments

Body weight and food intake were measured every week for all mice. All food was replaced weekly to avoid contamination. For collection of blood samples, mice were fasted for 6 h during the light phase period and blood was taken from the facial vein unless otherwise specified. After 6 h fasting, animals used in this study were euthanized by i.p. injection of pentobarbital (200 mg/kg). Several experiments with special treatments are described below.

1) Determination of sexual dimorphism in metabolic endotoxemia, low-grade chronic inflammation, and metabolic syndrome: Male (M) WT (n = 11) and female (F) (n = 11) mice were weaned and switched to western diet (WD) until the age of 20 weeks to induce severe WD-induced obesity and MS. Both groups were subjected to analysis of markers of metabolic endotoxemia (ME) (including LPS, LBP, sCD14, and intestinal permeability), systemic low-grade chronic inflammation (LGCI) (including TNF-α, IL-1β, IL-6, MCP-1, and IL-10), and metabolic syndrome (MS) (including weight gain, glucose tolerance test with area under the curve, insulin resistance index assessed by HOMA-IR, serum lipid profile including total cholesterol (TC), triglyceride (TG), low-density lipoprotein-C (LDL-C) and high-density lipoprotein-C (HDL-C) and atherogenic index, non-alcoholic fatty liver score and serum aspartate transaminase (AST) and alanine transaminase (ALT), and analysis of fecal microbiota by 16S rRNA gene sequencing and serum LPS levels by LAL assay. The mice were then euthanized and white adipose tissue (WAT), which includes visceral (vWAT), subcutaneous (sWAT), and epididymal (eWAT) fat pads, and liver weights were taken. A portion of the liver was stored in 10% formalin for histological analysis. Tissues were snap frozen in liquid nitrogen and then stored at − 80 °C for future analyses. 2) Determination of 17β-estradiol effects on gut microbiome, metabolic endotoxemia, low-grade chronic inflammation, and metabolic syndrome: Ten-week-old male (M), female (F), and ovariectomized (OVX) mice maintained on control diet were purchased from Charles River Laboratories and divided in to five groups (n = 5/group), and were fed WD and 17β-estradiol (E2) from week 11 to week 17. The five groups are (1) M, (2) F, (3) M+E2, (4) OVX, and (5) OVX+E2. E2 (Sigma, USA) was prepared and given to groups 3 and 5 in the drinking water as described previously [87]. E2 was dissolved in 95% ethanol (5 mg/mL). Solubilized E2 was added to the drinking water to produce concentration of 4000 ng E2/mL water with final ethanol concentration of 0.1%. Groups 1, 2, and 4 received a normal drinking water bottle with 0.1% ethanol. The water bottles were changed every week for all the groups. After 6 weeks, mice were subjected to analysis for markers of ME and systemic LGCI and MS as mentioned in section 1. The mice were then euthanized and WAT and liver weights were taken. A portion of the liver and duodenum was stored in 10% formalin for histological analysis. Tissues were snap frozen in liquid nitrogen and then stored at − 80 °C for future analyses. 3) Fecal microbiota transplantation (FMT): Mice from each group were individually housed for receiving antibiotic treatment to the end of the experiment. Fecal microbiota in place of cecal microbiota was transplanted for convenience and in order to minimize the number of animals used as donors. FMT with fecal content from donor male (M) mice that were fed WD for 10 weeks was performed on female 10-week-old mice [19]. Before the microbial transplantation, recipient mice (M → F) were treated with a 200 μL antibiotic cocktail (ampicillin, 1 g/l; metronidazole, 1 g/l; vancomycin, 0.5 g/l; neomycin, 0.5 g/l) (Sigma, USA) administrated by oral gavage once a day for 3 days. During the last 8 h of antibiotic treatment, mice were fed a WD to facilitate subsequent colonization. Fresh fecal pellets from donors were immediately weighed and placed into Ringer’s solution and then diluted to 10 mg/mL. Immediately after diluting the fecal materials, fecal solutions were gavaged (200 μL per mouse) to 4-h-fasted female recipients. Control groups of mice (males and females) were force-fed with 200 μL of transfer buffer alone to eliminate the effects of gavage per se. Two days later, these mice received another gavage to exclude possibility of any unsuccessful inoculation. We conducted this FMT procedure once a week for the next 3 weeks. After the first microbiota gavage, all mice were fed a WD for 20 weeks. Measurement of body composition (fat mass) was performed using nuclear magnetic resonance (NMR) technique (minispec Body Composition Analyzer based on Time Domain NMR) that provides noninvasive and rapid measurement without anesthetics. Fecal samples from these three groups (M/F/M → F; n = 5 per group) were collected after WD feeding for 20 weeks and three fecal samples from each group were subjected to 16S rRNA sequencing. 4) Antibiotic (ABX) treatment: 10-week-old male (n = 5) and female (n = 5) mice maintained on chow diet were switched to a WD until the age of 20 weeks to induce sexual dimorphism in MS. After collecting serum and stool for baseline measurements, both males and females receiving a WD started receiving a broad spectrum antibiotic cocktail (ABX) containing ampicillin (1 g/l), vancomycin (500 mg/l), neomycin sulfate (1 g/l) (added to the drinking water), and metronidazole (100 mg/kg) (orally gavaged every 12 h) for 6 weeks to deplete the gut microbiota [88]. Validation of successful depletion of gut microbiota after the antibiotic treatment was performed as described previously [88]. Briefly, bacterial cultivation (both aerobic and anaerobic bacteria) of feces was performed on day 24 of the antibiotic treatment mice as described previously [14]. The detection limit of the assay (successful depletion) was defined as 1 cfu/mg feces. In addition, the bacterial genomic DNA was extracted from fresh stool samples of these mice and 16S rRNA gene copies for all bacteria (Table S4) was measured with qPCR method as described below. Both bacterial cultivation (cfu/mg feces) and qPCR results (Ct values) were compared with results from pre-antibiotic treatment fecal samples to confirm a significant depletion of gut microbiota. Mice with antibiotic treatment were subjected to analysis for markers of ME and systemic LGCI and MS as mentioned in section 1. 5) Analysis of the effects of isoflavones (ISO) on gut microbiome, ME, LGCI, and MS: Ten-week-old male (M) mice were fed a WD for 4 months to induce MS and then divided into two groups: group 1: M, (n = 4), and group 2: M+ISO (n = 6). ISO such as genistein and daidzein (Cayman, USA) were supplemented at 0.1% in the WD, the M group was fed a WD, and the M+ISO group was fed a WD supplemented with ISO for the next 5 weeks. Mice were then subjected to analysis for markers of ME and systemic LGCI and MS as mentioned in section 1. The mice were then euthanized and white adipose tissue (WAT) and liver weights were taken. A portion of the liver and duodenum was stored in 10% formalin for histological analysis. Tissues were snap frozen in liquid nitrogen and then stored at − 80 °C for future analyses. 6) Analysis of the effects of intestinal alkaline phosphatase (IAP) inhibition on gut microbiome, ME, LGCI, and metabolic abnormalities: To determine the effects of inhibiting endogenous IAP activity, 12-week-old WT mice on a WD were divided into three groups ((male (M), female (F), and F+L-phe)) (n = 5 per group) and allowed to drink autoclaved water alone or water containing 10 mM L-phenylalanine (L-phe) (Sigma, USA). After 8 weeks, the serum and fecal samples were collected to analyze the markers of ME, LGCI, and MS.

Extraction of genomic DNA and profiling of the 16S rRNA gene by next generation

Sequencing

Fecal DNA extraction and 16S rRNA gene sequencing

Bacterial genomic DNA was extracted from fresh stool samples (100–180 mg) using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. To increase effectiveness, the lysis temperature was increased to 95 °C. Eluted DNA was treated with RNase and analyzed using a Nanodrop spectrophotometer (Biotek, Winooski, VT). Sample concentration and purity was determined by absorbance at 260 nm and the A260/A280 ratio, respectively. DNA samples packed with dry ice were shipped to APC Microbiome Institute (University College Cork, Cork, Ireland), and samples were sequenced as previously mentioned [37]. Briefly, V3–V4 amplicons for Illumina sequencing were generated according to the 16S metagenomic sequencing library protocol (Illumina). An initial PCR reaction utilized primers specific for amplification of the V3–V4 region of the 16S rRNA gene, (Forward primer 5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; reverse primer 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). PCR product clean-up and purification was achieved using the Agencourt AMPure XP system (Labplan, Dublin, Ireland). A second PCR incorporated a unique indexing primer pair for each sample (Illumina Nextera XT indexing primers, Illumina, Sweden). The products were again purified using the Agencourt AMPure XP system. Samples were quantified using the Qubit broad range DNA quantification assay kit (Bio-Sciences, Dublin, Ireland). Following quantification, samples were pooled in equimolar amounts (4 nM) and sequenced at Clinical Microbiomcs, Copenhagen, Denmark, using Illumina MiSeq 2 × 300 bp paired end sequencing.

a)Bioinformatics

Three hundred-base pair paired-end reads were assembled using FLASH with parameters of a minimum overlap of 20 bp and a maximum overlap of 120 bp [89]. The QIIME suite of tools, v1.8.0, was used for further processing of paired-end reads, including quality filtering based on a quality score of > 25 and removal of mismatched barcodes and sequences below length thresholds [90]. Denoising, chimera detection, and operational taxonomic unit (OTU) grouping were performed in QIIME using USEARCH v7 [91]. Taxonomic ranks were assigned by alignment of OTUs using PyNAST to the SILVA SSURef database release 111 [92]. Generation of α and β diversities and analysis and visualization of principal coordinate analysis (PCoA) plots were performed using PAST and XLSTAT software. The α-diversity of each group was calculated based on the annotated data using the diversity indices of the PAST version 2.17 software program [93]. Based on a non-parametric two-sample t-test using the default number of Monte Carlo permutations (999), comparative analyses of the group-specific α-diversity indices were performed. Ordinations are the dimensional-reduction techniques which are commonly used to visualize complex relationships between communities between groups (β-diversity). Dimensional reduction of the Bray-Curtis distance between microbiome samples using PCoA ordination method (PAST software) was done and significant differences among groups were tested with permutational multivariate analysis of variance (PERMANOVA), a multivariate non-parametric one-way ANOVA, which utilizes the sample-to-sample Bray-Curtis distance matrix directly. Taxa which were primarily responsible for an observed difference between groups were identified by SIMPER (similarity percentage analysis) method and their contribution to groups (between and within groups) were analyzed using the PCA variance-covariance type ordination (PAST software) method. Differential abundance analysis (non-parametric ANOVA with Benjamini-Hochberg FDR-corrected P values < 0.05) was performed on the RA of microbiota data at different levels of taxonomy to identify taxa with FDR-corrected P values < 0.05 (XLSTAT software; Addinsoft, USA) [94] and then their RA (normalized to percentage) were shown by a heat map with hierarchal clustering (HCN) analysis [95] using GraphPad Prism version 7.01 (La Jolla, CA). Linear discriminant analysis (LDA) effect size (LEfSe) is a biomarker discovery and explanation tool for high-dimensional data. It couples statistical significance with biological consistency and effect size estimation [96]. Microbiota-based biomarker discoveries were done with LEfSe using the online galaxy server (https://huttenhower.sph.harvard.edu/galaxy/), and the LDA scores derived from LEfSe analysis [96] were used to show the relationship between taxon using a cladogram (circular hierarchical tree) of significantly increased or decreased bacterial taxa in the gut microbiota between groups. Levels of the cladogram represent, from the inner to outer rings, phylum, class, order, family, and genus. Color codes indicate the groups, and letters indicate the taxa that contribute to the uniqueness of the corresponding groups at an LDA of > 2.0. Unweighted pair-group method with arithmetic means (UPGMA) hierarchical clustering analysis diagram based on Bray-Curtis distance matrix was obtained using PAST version 3.11. Class trees were used to demonstrate similarity between samples, the clustering tree branch length was a measure of the cluster effect.

b)Putative metagenome identification

Microbial functions were predicted using 16S ribosomal RNA sequencing and phylogenetic reconstruction of unobserved states (PICRUSt) software (version 1.0.0) as described [29]. The predicted genes and functions were aligned to the KEGG database (version 66.1, May 1, 2013). PCA and PERMANOVA statistics were applied to check whether the groups were clustered according to predicted gene enrichments for microbial functions. LEfSe analysis [96] was utilized to determine significant putative KEGG orthologs and pathway analyses [96].

Lipopolysaccharide (LPS) concentration

Serum LPS concentrations were measured with a Toxin Sensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript, Piscataway, NJ), following the manufacturer’s instructions [15]. Briefly, serum samples were diluted 10- to 50-fold with endotoxin-free water, adjusted to the recommended pH, and heated for 10 min at 70 °C to minimize inhibition or enhancement by contaminating proteins. LAL reagents were added to serum and incubated at 37 °C for 45 min, and the absorbance was read at 545 nm. All samples were validated for recovery and internal coefficient variation using known amounts of LPS.

Intestinal permeability

Intestinal permeability was determined as previously described [15]. Briefly, mice were gavaged with phosphate buffered saline (PBS, pH 7.2) containing 600 mg/kg body weight FITC-dextran (40 kDa, Sigma-Aldrich, USA). Blood samples (120 μL) were collected after 90 min. Serum was diluted with an equal volume of PBS, and fluorescence intensity was measured using a fluorospectrophotometer (excitation wavelength 480 nm and emission wavelength 520 nm; Perkin-Elmer, Waltham, MA). Serum FITC-dextran concentrations were calculated from a standard curve of serially diluted FITC-dextran in PBS.

Measurement of intestinal alkaline phosphatase (IAP) level and activity

Small intestinal IAP specific activity (as it relates to protein) was measured as previously described [15] and expressed as picomoles pNPP hydrolyzed/min/μg of protein. Briefly, thoroughly washed duodenal tissues were homogenized with lysis buffer (150 mM NaCl, 10 mM Tris·HCl, pH 7.5, 1% sodium deoxycholate, 1% Nonidet P-40, 10 mM EDTA, 0.1% SDS, including protease inhibitor mixture; Sigma) followed by incubation on ice for 30 min. Thereafter, the homogenates were centrifuged twice at 4 °C at 15,000g for 15 min, and the supernatants were collected to determine IAP activity as well as protein concentration. The Coomassie Blue Protein Assay (Bradford) kit from Fisher Scientific was used for protein quantification. For IAP assay, 25 μL of supernatant was mixed with 175 μL phosphatase assay reagent containing 5 mM of p-nitrophenyl phosphate (pNPP) followed by determining optical density at 405 nm. The specific activity of the enzyme was expressed as picomoles pNPP hydrolyzed/min/μg of protein. Protein concentration in a specific sample was determined using the protein assay reagents from Fisher Scientific.

Cell culture experiments

The human colon carcinoma cell line (Caco-2) was obtained from American Type Culture Collection (ATCC) (Rockville, MD) and cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning Inc., NY, USA) containing 4.5 g/l glucose, 4 mmol/l l-glutamine, and 1 mmol/l sodium pyruvate, and supplemented with 10% fetal bovine serum (Cell Applications, Inc., San Diego, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, NY, USA) in a humidified atmosphere of 5% CO 2 at 37 °C. They were routinely subcultured when they were about 80% confluent. The culture medium was changed every other day. Cells were always > 90% viable, as shown by trypan blue (Invitrogen, Carlsbad, CA, USA) exclusion. Cells were passaged every 3–4 days by treatment with 0.1% trypsin (Gibco) and 0.04% ethylenediaminetetraacetic acid (EDTA) and then plated at a density of 1.3–2 × 104 cells/cm2. Cells at passage number 17 were used for the experiments. All assays were done using only differentiated Caco-2. Cells were seeded in to six-well plates at 2 × 104 cells per well and treated with vehicle (ethanol or DMSO) or 10 nM 17β-estradiol (E2) for 24, 48, and 72 h or each 25 μM genistein (G) or daidzein (D) or G+D mixture for 72 h. In a subset of experiments, Caco-2 cells were pre-treated with L-phenylalanine (10 mM) for 24 h and then they were treated with either vehicle or each 25 μM G+D mixture or 10 nM E2 for 72 h. Medium was removed and cells were washed twice with ice-cold PBS, scraped, lysed in Trizol (Invitrogen), and stored at − 80 °C until mRNA was isolated or homogenized with 200 μL radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail (Sigma), incubated on ice for 30 min, centrifuged at 14,000g for 10 min at 4 °C and the supernatant was collected and stored at − 80 °C for western blotting analysis.

Western blotting analysis of IAP

Western blotting on tissues and Caco-2 cell lysates was performed as previously described [14]. After thawed, the protein samples derived from Caco-2 cells, the homogenates were centrifuged at 15,000g at 4 °C for 15 min, and the supernatants were collected. The duodenum section of small intestinal tissues was harvested and cut open longitudinally and luminal contents were removed. The tissues were washed with PBS and homogenized with liquid nitrogen, and homogenates were mixed with RIPA buffer, incubated on ice for 30 min, and centrifuged at 14,000g for 10 min at 4 °C, and the supernatant was collected. Protein concentration of Caco-2 and tissue homogenates was quantified by the Coomassie blue protein assay (Thermo Scientific, Rockford, IL, USA) using bovine serum albumin (BSA) as the standard. Proteins (30 μg) were resolved on SDS-PAGE gels and transferred onto nitrocellulose membranes (Osmonics, Minnetonka, MN, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then probed with IAP primary antibodies (GTX27322, GeneTex, San Antonio, TX, USA) in 5% non-fat dry milk in TBS-T at 4 °C overnight. After washing three times in TBS-T, the blots were further incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature (Santa Cruz Biotechnology, Santa Cruz, CA). Chemiluminescence was detected with Pierce ECL western blotting substrate (Thermo Scientific, Rockford, IL, USA) and visualized by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Immunohistochemical analysis of IAP

Formalin-fixed duodenal tissues and IAP primary antibodies (GTX27322, GeneTex) [14] were given to MGH core (Boston, MA). Prepared IHC slides were analyzed under light microscope and images of IAP staining and localization was taken by using × 20 magnifications. All pictures were taken with the same exposure conditions without autoscaling.

RV coefficient

The RV coefficient was calculated between the microbial genera (FDR-corrected P value < 0.05) and the host parameters (markers of ME, LGCI and MS). The RV coefficient is a multivariate generalization of the Pearson correlation coefficient [97].

Correlation network analysis

Network-based analytical approaches have the potential to help disentangle complex host-microbe interactions [98]. Pairwise correlations between each microbiota (genera that are present at < 0.1% relative abundance in > 75% samples have been removed to avoid detecting spurious correlations among low-abundance OTUs) and host parameter (markers of ME, LGCI, and MS) were calculated using Spearman’s nonparametric rank correlation coefficient [99]. Using those significant (P < 0.05) correlation coefficients, a correlation network (Fruchterman Reingold and label adjust layout) was built where nodes represent either a microbiota or a host parameter. For each microbiota and a host parameter, an undirected edge was added between the corresponding nodes in the correlation network. Edges (light black links indicate positive and blue links indicate negative associations) represent statistically significant correlations (P < 0.05). Correlations were calculated using the PAST software version 2.17 and the network was visualized in Gephi Graph Visualization and Manipulation software version 0.9.2 [100]. Nodes were colored based on “data type” and sized based on “betweeness centrality (BC).” BC is a network centrality measure that quantifies the influence of a node in connecting other nodes in a network. It represents the fraction of all shortest paths in the network that pass through a given node. The nodes with the highest BC are usually known as highly central or hubs. A “module or component” in the network is a set of nodes connected to each other by many links, while connected by few links to nodes of other groups, so modules are elementary units of any biological network, and their identification and characterization provides us with more information about the local interaction patterns in the network and their contribution to the overall structure, connectivity, and function of the network. Modules are biologically important when considered as isolated, taxonomic, evolutionary, or functional modules. High modularity indicates that the network has dense connections within certain groups of nodes and sparse connections between these groups.

Multivariate statistical analysis

Partial least square regression (PLS-R) was used to associate the microbial composition to host parameters including jackknife-based variable selection [98]. PLS-R is recommended in cases of regression where the number of explanatory variables is high, and where it is likely that the explanatory variables are correlated. Leave one-out cross-validation (LOO-CV) was applied. The Q2 cumulated index (Q2 cum ) measures the global goodness of fit and the predictive quality of the models. Q2 cum is also used to test the validity of the model against over-fitting. The cumulated R2Y and R2X cum that corresponds to the correlations between the explanatory (X) and dependent (Y) variables with the components are very close to one with two components in all the models. This indicates that the two components generated by the PLR-R summarize well both the Xs and the Ys. The results are also presented in PLS scatter plots for subject clustering and variables. The R2 (coefficient of determination) indicates the % of variability of the dependent variable (Y) which is explained by the explanatory variables (X). Parameters (variable importance in the projection values 1 or > 1.0) contributing to the multivariate PLS models were compared with the corresponding identified modules (Fig. 7b–d) in the correlation networks. All analyses were performed using precise algorithm in the XLSTAT software version 2017.6.

Statistical analysis

Data was shown as mean ± standard error of mean (SEM). Box-plots (box showing the median, and the 25th and 75th percentiles, and the whiskers of the graph show the largest and smallest values) were also used to express the data. Unpaired Student’s t test was performed for experiments having only two groups. Either ordinary or repeated measures one-way or two-way analysis of variance (ANOVA) with Tukey’s or Sidak’s multiple comparisons post-test were used for experiments having more than two groups. If unequal variance was detected, data were analyzed using non-parametric tests. Differences were considered significant at P < 0.05. Statistical analyses, including heat-map preparation, were performed using GraphPad Prism version 7.01 (GraphPad Software, La Jolla, CA). Differential expression analysis on 16S sequencing data was conducted using XLSTAT software program [94]. Multivariate statistical analyses and power analyses (alpha = 0.05; effect size = 0.8) were conducted using PAST (version 2.17) [93] and XLSTAT (version 2017.6) software products.