All regents and oligoes used in this study were listed in supplementary Table S1.
Four-to six-week-old male or female C57BL/6 mice were obtained from Nanjing Animal Center, Nanjing, China; AhR KO mice were from the Third Military Medical University, Chongqing, China; PXR KO mice and CAR KO mice from Chinese Academy of Inspection and Quarantine ,Tianjin, China; TLR4 KO mice were from Shanghai Model Organisms Center, Shanghai, China. All experimental litters were bred and maintained under specific pathogen-free conditions in Nankai University. Experiments were carried out using age- and gender- matched mice. All the procedures were conducted according to the Institutional Animal Care and Use Committee of the Model Animal Research Center. Animal experiments were approved by the Institute’s Animal Ethics Committee of Nankai University. All experimental variables such as husbandry, parental genotypes and environmental influences were carefully controlled.
C57BL/6 germ-free (GF) mice were generated by Beijing Animal Center. All experiments in GF mice were performed in Institute of Laboratory Animal Science，Chinese Academy of Medical Sciences (CAMS) Comparative Medical Center, Peking Union Medical College (PUMC), Beijing 100021, China.
Reg4 deficient (KO) mice were generated by CRISPR/Cas-mediated genome engineering as described before(Qi et al, 2020).
Human REG4 transgenic mice ( huREG4IEC tg) mice were prepared by Nanjing Animal Center. HD5 promoter, which may specifically promote the REG4 expression in gut Paneth cells, was conjugated into Pinsulator–pHD5-promoter-CDS-poly plasmid. The fragment of REG4-CDS and polyA were cloned into HD5 promoter-Pinsulator. This conjugation was demonstrated using primers (REG4-HD5-tF: CATCCAACTCCAGGACGGAGTC and REG4-HD5-tR: CACCTGTAACATTGGCACTTTG) and sequencing using the primers (Promoter-cx tF: GTTTGCTGGGTCAGAACTGA and Promoter-cx tR: GTAATTTAGGTGCGTACAGCC). HuREG4IEC tg mice were identified using the following primers (HD5-Test-Tf:CTGTCTCAGGTCTTCTCCCAG and REG4 -PA-F: GATCTTTTTCCCTCTGCCAAA, which produced 323bp band in tg mice and no band in WT mice). Human REG4 tg mice or WT siblings were from a cross between WT mice and human REG4 tg mice (heterozygous mice).
For human serum collection, 208 adult participants, among which 42 with BMI<18.5 kg/cm2, 41 with BMI 18.5-24 kg/cm2, 43 with BMI 24-27 kg/cm2, 42 with BMI 27-30 kg/cm2 and 40 with BMI ≥30 kg/cm2 were selectively recruited. More than half (54.3%) were males and the mean age was 42 years (SD=13 years). The mean BMI was 25.05±5.07 kg/cm2. All participants were free of acute stress conditions such as fever and diarrhea. Height and weight were measured to the nearest 0.1cm and 0.1 kg without shoes or heavy clothing using a calibrated stadiometer (GL-310, Seoul, Korea). Participants were instructed to fast for ≥ 12 hours before blood sampling the next morning. This study was conducted with approval from the Institutional Review Board at Nankai University, Tianjin Union Medical Center and Tianjin First Central Hospital. Participants were recruited from the health screening centers of Tianjin Union Medical Center and Tianjin First Central Hospital. All participants provided written informed consent.
For high-fat diet model, 6-8 week old male mice and their control littermates were fed using high-fat diet (D12492, protein 26.2%, carbohydrate, 26.3% and Fat 34.9%) and control diets (D12450B(60% of calories may be derived from fat), which was from Research Diets, Inc..(NJ, USA).
For microbiota transplantation, 6- to 8-week-old mice were treated with pan-antibiotics (ampicillin (A, 1 g/L, Sigma), vancomycine (V, 0.5g/L), neomycin sulfate (N, 1 g/L), and metronidazole (M, 1g/L)) via the drinking water. Water containing antibiotics was exchanged every three days. To confirm the elimination of bacteria, stool was collected from antibiotic-treated and untreated mice and cultured in anaerobic and aerobic condition. The bacteria were counted under microscope. Then cecal contents from detergent treated mice or bacteria (1×109) were suspended in 1 ml PBS with 30% glycerol. Mice (4 weeks old) were removed from the isolator and were orally administered 200 ml of fecal suspension or bacteria made using glycerol stocks.
For lactobacillus transplantation, mice were treated with pan-antibiotics via the drinking water for two weeks. After confirming the elimination of bacteria, 1×109 CFU/0.2ml PBS Lactobacillus was infused into mice with intraperitoneal injection with 2mg/kg LPS (once/ week for three weeks). After infusing lactobacillus, mice were fed with enriched (0.48%) tryptophan diets.
For in vivo administration of IAA plus LPS, mice were randomly divided into 4 groups, normal group with 0.2 ml PBS only, IAA group i.p with 500 mg/kg IAA diluted in DMSO, LPS group with 2 mg/kg O111:B4 in 0.2ml PBS, and IAA plus LPS group with 500mg/kg IAA and 2 mg/kg O111:B4. After administration for 24 hrs, spleen and PP tissues were separated for further analyses.
Ex vivo stimulation
For Ex vivo stimulation, mice spleen cells were collected. 5×106 cells per well were seeded into 24-wells plate, and stimulated with IAA(100μM), LPS(100ng/ml), or IAA(100μM) plus LPS(100ng/ml) with or without PXR inhibitor(10uM), AHR inhibitor (10uM), or NF-κB inhibitor (10uM) for 24hrs.
For WEHI231 B cells stimulation, WEHI231 B cells were seeded in 24-wells plate. The cells were then stimulated with IAA and LPS with or without PXR inhibitor or NF-κB inhibitor for 3hrs or 6 hrs, and harvested for WB, CHIP-SEQ and CHIP-PCR analyses.
For glucose and insulin tolerance, after 5 hrs fasting, baseline blood glucose levels were measured using a Nova Max Plus GlucoseMeter. Mice were then injected intraperitoneally with 2 g glucose per kg body weight in sterile PBS or with 0.5U insulin per kg body weight (Sigma, St. Louis, Missouri), and blood glucose levels were measured at different times after injection.
Gut microbiota analyses
Previous protocol was used in gut microbiota analyses (Qi et al, 2019). Brefiely, Gut microbiota was analyzed by Majorbio Biotechnology Company (Shanghai, China) using primers that target to the V3-V4 regions of 16S rRNA. Once PCR for each sample was completed, the amplicons were purified using the QIAquick PCR purification kit (Qiagen Valencia, CA), quantified, normalized, and then pooled in preparation for emulsion PCR followed by sequencing using Titanium chemistry (Roche, Basel Switzerland) according to the manufacturer’s protocol. Operational Taxonomic Unit (OTU) analysis was performed as follows: sequences were processed (trimmed) using the Mothur software and subsequently clustered at 97% sequence identity using cd-hit to generate OTUs. The OTU memberships of the sequences were used to construct a sample-OTU count matrix. The samples were clustered at genus and OTU levels using the sample-genus and sample-OTU count matrices respectively. For each clustering, Morisita-Horn dissimilarity was used to compute a sample distance matrix from the initial count matrix, and the distance matrix was subsequently used to generate a hierarchical clustering using Ward’s minimum variance method. The Wilcoxon Rank Sum test was used to identify OTUs that had differential abundance in the different sample groups.
For lactobacillus isolation, 100 mg fresh stool samples were collected and diluted in 2 ml BPS solution, and cultured on Rogosa SL selective medium (Sigma-Aldrich) for Lactobacillus enumeration, and then colonies were identified and purified using 16s ribosomal DNA sequence analyses for the speciation of colonial genotype. The lactobacilli were cultured in deMan, Rogosa, Sharpe (MRS; 3 M Health Care, St. Paul, MN) media and also grown on MRS agar containing either 10% sucrose. Anaerobic conditions were generated with sachets of AnaeroPack-Anaero (Mitsubishi Gas Chemical, Japan) in an air-tight jar. At 24 h of cultivation in liquid media, isolated lactobacillus Ruteri could reach 1 × 109 CFU/ml).
IaaM gene deletion in lactobacillus
For lactobacillus iaaM gene deletion, the upstream and downstream fragments of the iaaM gene of lactobacillus were first amplified. The purified upstream and downstream homologous fragments were inserted into the Xoh I, Pem I and Sac I, Bgl II digestion sites of the pNZ5319 plasmid, respectively. Receptive cells of lactobacillus were prepared and the recombinant pNZ5319 plasmid was electrotransferred into the receptive cells of lactobacillus with the electrotransfer parameters of 1.7 kV (2 mm electrode cup), 200 Ω resistance and 25 μF capacitance. Single colone of chloramphenicol-resistant lactobacillus were selected, and the iaaM-up-F/R, iaaM-down-F/R, CM-F/R primers were used to validate the strains with single exchange. The single-exchange strains were cultured for 3 generations per day at 30°C, and the suspensions were taken at 40 generations for double-exchange strain screening. The colonies that could grow normally on plates containing chloramphenicol resistance but not on solid plates containing erythromycin resistance were selected. The screened double exchangers were verified using iaaM-F/R PCR. Double-exchanger positive bacteria were prepared as receptor cells and electrotransfected with pNZTs-Cre plasmid for 3 generations per day to about 10 generations, eliminating the chloramphenicol resistance gene from the genome and verified using CM-F/R PCR. The heat-sensitive plasmid pNZTs-Cre was eliminated by incubation in an incubator at 42°C for 3-5h.
LC-MS (Liquid chromatography-mass spectrometry)/MS
The samples were thawed at room temperature. All samples were extracted with 300L of methanol, and 10L of internal stardard (2.9 mg/mL, DL-O-Chlorophenylalanine) was added. The samples were vortexed for 30 s, and centrifuged at 12000 rpm and 4oC for 15 min. Waters ACQUITY UPLC HSS T3 column (2.1 mm100mm, 1.8 M) was used. Chromatographic separation conditions are : column temperature, 40oC; Mobile phase A, water+0.1% formic acid; Mobile phage B, acetonitrile+0.1% formic acid; Flow rate, 0.35 mL/min; injection volume, 6L. MS parameters used for ESI+ were: capillary voltage, 1.4 kV; Sampling cone, 40 V; Source temperature, 120 °C; Desolvation temperature, 350°C; Cone gas flow, 50 L/h; Desolvation gas flow, 600 L/h; Collision energy, 10-40V; Ion energy, 1V; Scan time, 0.03 s; Inter scan time, 0.02 s; scan range, 50-1500 m/z. MS parameters used for ESI- were: capillary voltage, 1.3 kV; Sampling cone, 23V; Source temperature, 120 °C; Desolvation temperature, 350 °C; Cone gas flow, 50 L/h; Desolvation gas flow, 600 L/h; Collision energy, 10-40V; Ion energy, 1V; Scan time, 0.03 s; Inter scan time, 0.02 s; and scan range, 50-1500 m/z. Analysis platform was performed by ACQUITYTM UPLC-QTOF.
The data was performed by feature extraction and preprocessed with Masslynx 4.1 software (Waters), and then normalized and edited into two-dimensional data matrix by excel 2010 software, including retention time(RT), mass, observations(samples) and peak intensity. The data after editing were performed Multivariate Analysis (MVA) using SIMCA-P 13.0 software (Umetrics AB, Umea, Sweden).
Cell isolation and flow cytometry
Previous reported protocol was used in cell isolation and flow cytometry(Cao et al, 2016). Briefly, single-cell suspensions of Peyer’ s patches (PP) and spleen of mice were prepared by mashing in a cell strainer (70 mm), stained and analyzed by flow cytometry. For the staining of lamina propria (LP) lymphocytes, gut were isolated, cleaned by shaking in ice-cold PBS four times before tissue was cut into 1 cm pieces. The epithelial cells were removed by incubating the tissue in HBSS with 2 mM EDTA for 30 min at 37oC with shaking. The LP cells were isolated by incubating the tissues in digestion buffer (DMEM, 5% fetal bovine serum, 1 mg/ ml Collagenase IV (Sigma-Aldrich) and DNase I (Sigma-Aldrich) for 40 min. The digested tissues were then ﬁltered through a 40-mm ﬁlter. Cells were resuspended in 10 ml of the 40% fraction of a 40: 80 Percoll gradient and overlaid on 5 ml of the 80% fraction in a 15 ml Falcon tube. Percoll gradient separation was performed by centrifugation for 20 min at 1,800 rpm at room temperature. LP cells were collected at the interphase of the Percoll gradient, washed and resuspended in medium, and then stained and analyzed by flow cytometry. Dead cells were eliminated through 7-AAD staining. For the staining of immune cells in adipose tissues, adipose tissues first were cut into smaller pieces and then digested in digestion buffer (20l / ml Collagenase I (Sigma-Aldrich) )for 35 min.. The digested tissues were then ﬁltered through a 40-mm ﬁlter.
For analysis of different immune cell populations, the cells were washed with staining buffer containing 2% FBS, 1 mM EDTA and 0.09% NaN3 and surface staining was performed with APC, FITC, PercP, BV 605 or PE-labeled anti-CD4, CD19, p35, EPI3, CD11c, MHCII, F4/80, CD11b, Ly6C and CD45 antibodies and analyzed using FACScan flow cytometry (Su et al, 2014).
For intracellular staining, the cells were cultured and stimulated with 50ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μg/ml ionomycin (Sigma) in the presence of GolgiStop (10 ng/ml, BD Biosciences). After incubation for 6 hrs, cells were washed in PBS, and then fixed in Cytofix/Cytoperm, permeabilized with Perm/Wash buffer (BD Biosciences), and stained with FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5- or PE/cy7-conjugated antibodies. Meanwhile, dead cells were eliminated through 7-AAD staining.
For absolute cell number count, spleen tissue and whole PP nodes of mice were weighed and single-cell suspensions were prepared for flow staining analysis. The total number of cells per gram of spleen or PP node was counted and then multiplied by the proportion of positive cells to obtain the absolute number of cells.
H & E staining
For hematoxylin/eosin (H&E) staining, previously reported methods were used in this experiment (Su et al, 2014). Briefly, adipose tissues were fixed in 4% (w/v) paraformaldehyde buffered saline and embedded in paraffin, 5 µm sections colon sections were cut and stained with H&E.
For immune staining, previous reported protocol was used(Shang et al, 2019). Briefly, colon tissues or spleen were embedded in OCT compound (Tissue-Tek, Torrance, CA) and frozen over liquid nitrogen. 5-μm-thick sections were prepared from frozen tissue and fixed in acetone (−20°C) for 10 min. After rehydration in PBS for 5 min and further washing in PBS, tissue sections were blocked with 1% (w/v) BSA and 0.2% (w/v) milk powder in PBS (PBS-BB). The primary antibody was added in PBS-BB and incubated overnight at 4°C. After washing (three times, 5 min each), tissue was detected with DAB kit or fluorescence labeled second antibody. Nuclei were stained by DAPI.
Immunoprecipitation and immunoblot
Immunoprecipitation and immunoblot were performed according to previous methods (Su et al, 2014). The cells were lysed with cell lysis buffer (Cell Signaling Technology), which was supplemented with a protease inhibitor 'cocktail' (Calbiochem). The protein concentrations of the extracts were measured using a bicinchoninic acid assay (Pierce). For the immunoblot, hybridizations with primary antibodies were conducted for 1 h at room temperature in blocking buffer. The protein-antibody complexes were detected using peroxidase-conjugated secondary antibodies (Boehringer Mannheim) and enhanced chemiluminescence (Amersham).
CHIP-seq and CHIP-PCR
Chromatin immunoprecipitation (ChIP)-PCR was performed using EZ-ChIP™ Chromatin Immunoprecipitation Kit (Millipore) according to our previously reported methods(Gao et al, 2018). Briefly, the cells were crosslinked with 1% paraformaldehyde and incubated with rotation at room temperature. Crosslinking was stopped after 10 min with glycine to a final concentration of 0.125M and incubated 5 min further with rotation. Cells were washed with ice cold PBS (containing 1% PMSF) 3 times and immediately resuspended in SDS lysis buffer (containing 1% PMSF). Cell lysates were sonicated for 40 cycles of 30 sec on and 30 sec off in 10 cycle increments using a Biorupter (Diadenode) on ice. After pelleting debris, protein G agarose was added and incubated for 1 hour at 4°C with rotation for preclearing. For immunoprecipitation, precleared cell lysate was incubated with the indicated antibodies overnight with the rotation at 4°C, and protein G agarose was added for the final 2 hrs of incubation. Beads were washed with low salt, high salt, LiCl wash buffer, and chromatin immunocomplex was eluted using elution buffer through incubating at room temperature for 15 minutes. Reverse crosslinks of protein/DNA complexes to free DNA were realized through adding 5 M NaCl and incubating at 65°C overnight. CHIP-sequence and qPCR was performed on DNA purified after treatment with RNase (30 min, 37°C) and proteinase K (2h, 55°C) after reversal of crosslinks.
RT-PCR and qRT-PCR
RT-PCR and qRT-PCR were performed according to our previous methods (Su et al, 2014). Total RNA was extracted from the cells, tissues and organs using TRIzol reagent (Invitrogen). First-strand cDNA was generated from total RNA using oligo-dT primers and reverse transcriptase (Invitrogen Corp). The PCR products were visualized on 1.0% (wt/vol) agarose gels. Quantitative real-time PCR (qRT-PCR) was conducted using QuantiTect SYBR Green PCR Master Mix (Qiagen) and specific primers in an ABI Prism 7000 analyzer (Applied Biosystems). GAPDH mRNA expression was detected in each experimental sample as an endogenous control. All reactions were run in triplicate..
The concentration of cytokines and IAA in the tissue, cell culture supernatants and sera was measured using ELISA kit.
Student’s t-test, one-way analysis of variance and the Mann–Whitney U test were used to determine significance. A 95% confidence interval was considered significant and was defined as p < 0.05.