Age, BMI and glycaemic status of the different cohorts can be found in Figure S1a.
Cohort 1 and 3
In cohort 1, a group of 154 [84 visceral (VsW) and 70 subcutaneous (ScW) adipose tissues] from participants with a wide range of adiposity (BMI between 20 and 68 kg/m2), were analysed. In cohort 3, 46 VAT and 36 SAT samples from morbidly obese subjects (BMI > 35 kg/m2), were analysed. Altogether these subjects were recruited at the Endocrinology Service of the Hospital of Girona “Dr Josep Trueta”. All subjects were of Caucasian origin and reported that their body weight had been stable for at least three months before the study. Subjects were studied in the post-absorptive state. They had no systemic disease other than obesity and all were free of any infections in the previous month before the study. Liver diseases (specifically tumoral disease and HCV infection) and thyroid dysfunction were specifically excluded by biochemical work-up. All subjects gave written informed consent, validated and approved by the ethical committee of the Hospital of Girona “Dr Josep Trueta”, after the purpose of the study was explained to them. Samples included in this study were partially provided by the FATBANK platform promoted by the CIBEROBN and coordinated by the IDIBGI Biobank (Biobanc IDIBGI, B.0000872), integrated in the Spanish National Biobanks Network and they were processed following standard operating procedures with the appropriate approval of the Ethics, External Scientific and FATBANK Internal Scientific Committees. AT samples were obtained from SAT and VAT depots during elective surgical procedures (cholecystectomy, surgery of abdominal hernia and gastric by-pass surgery). Samples of AT were immediately transported to the laboratory (5-10 min). The handling of tissue was carried out under strictly aseptic conditions. AT samples were washed in PBS, cut off with forceps and scalpel into small pieces (100 mg), and immediately flash-frozen in liquid nitrogen before stored at -80ºC. Serum glucose concentrations were measured in duplicate by the glucose oxidase method using a Beckman glucose analyser II (Beckman Instruments, Brea, CA, United States). Intra-assay and inter-assay coefficients of variation were less than 4% for all these tests. Total serum triglycerides were measured by an enzymatic, colorimetric method with glycerol phosphate oxidase and peroxidase (Cobas TRIGL) using a Roche Hitachi Cobas c 711 instrument.
ScW and VsW (omental) biopsy samples were obtained from severely obese (BMI >35 kg/m2) and lean subjects (BMI ≤25 kg/m2) who were undergoing elective surgery. Obese patients were candidate for bariatric surgery and the studies were conducted in Paris France and Spain in accordance with the Helsinki Declaration and approved by the Ethics Committee of Clinical Research (CPP Ile-de-France 1, Fibrota study N° clinical trial NCT01655017) and University Hospital of Girona Dr. Josep Tructa). All patients were characterized for detailed corpulence and metabolic phenotyping as described in16. Blood sampling were performed one month before the surgery at the fasting state. Paired AT samples (visceral and subcutaneous fat) were obtained at the time of the surgery by the same surgeon. Signed informed consents were obtained in all lean and obese individuals in agreement with ethic regulation.
Male C57Bl6/J mice were purchased from Charles River. Pepd KO mice were generated by the Wellcome Trust Sanger Institute Mouse Genetics Project on a C57Bl6/J background by mating heterozygous mice. Wild type and leptin deficient mice, LepOb/Ob , were on a C57BL/6 background. Studies were conducted in 8-28 weeks old mice using littermate controls. This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). Mice were housed 3–4 per cage in a temperature-controlled room (21°C) with a 12 hr light/dark cycle, with ‘lights on’ corresponding to six am. Animals had ad-libitum access to food and water. A standard chow diet (DS-105, Safe Diets) was administered to all animals from weaning, consisting of 64.3% carbohydrate, 22.4% protein and 13.3% lipid of total calories. Only male mice were used for in vivo experiments and preparation of BMDMs.
Generation of Bone Marrow Chimeras
C57BLl6/J mice at 10 weeks of age received a sub-lethal dose of whole-body irradiation (9 Gy). The day after irradiation, donor pepd KO mice were culled, and their femurs and tibias were removed aseptically. Marrow cavities were flushed in RPMI medium, and single-cell suspensions were prepared. The irradiated recipients received 1 × 107 bone marrow cells in 0.1 ml of PBS by tail vein injection. During 4 weeks after BMT, Bactrim (Roche) was added to drinking water. After 2 additional weeks, mice were switched to HFD 58%. Mice were culled 16 weeks later to collect blood and tissues.
Diets and Pharmacological challenges
Diets for animal studies included standard chow (10% calories from lipid), HFD 45% (D12451, Research Diets, 45% calories from lipid) and HFD 58% (D12331, Research Diets, 58% calories from lipid). Standard chow or HFD was provided ad libitum to animals from 8weeks old until indicated. Regarding PEPD pharmacological inhibition in vivo, 8 weeks-old C57BLl6/J mice were first fed with chow for 10 weeks. CBZ-Pro supplemented pellets were prepared fresh every other day in house by spraying homogeneously a solution of CBZ-Proline (2mM) in 70% ethanol solution. Mice consumed (when pellets were dry) an approximatively daily dose of 60mg/kg of CBZ-Proline, as previously used in other murine studies7 , treatment lasted for 6 weeks, control mice were offered regular pellets. CBZ-Proline does not alter viability or promote toxic effects in mice 7. In our experimental conditions, CBZ-Pro-treated mice did not show evidence of hepatotoxicity/liver damage (alanine (ALAT) or aspartate (ASAT) transaminase levels) (Extended data Figure 2a).
Fat and lean masses were calculated by time-domain nuclear magnetic resonance (TD-NMR) by using a minispec Live Mice Analyzer LF50 (Bruker).
Glucose and Insulin Tolerance Tests
For glucose tolerance test, mice were fasted for overnight with free access to drinking water. Glucose was administered intraperitoneally (2 g/kg), and blood glucose levels were monitored from the tip of the tail with a glucometer. For insulin tolerance tests, insulin was administered intraperitoneally (0.75mU/g), and blood glucose was measured at various times after injection.
Triglycerides were measured on the Dimension RXL analyzer (Siemens Healthcare). Free fatty acids were measured using the Roche Free Fatty Acid Kit (half-micro test) (kit code 11383175001). Insulin was measured using electrochemical luminescence immunoassay on the MesoScale Discovery immunoassay platform.
Explants for conditioned medium
Approximately 100 mg of freshly dissected GnW cut into fine pieces from 30-week-old mice in chow and HFD conditions were incubated for 6h hour at 37oC in 5% CO2 in DMEM with 5% heat inactivated FBS, 20 mM HEPES, 100 units/mL penicillin, 100 μg/mL streptomycin, and 20 mM L-glutamine) (1 mL media per 100 mg of tissue).
Magnetic-activated cell sorting
ScW, GnW and BAT from 10-12 weeks old C57BL/6 mice were dissociated by collagenase treatment isolating unilocular adipocytes from the stromavascular fraction (SVF). SVF was resuspended in MACS buffer (PBS, 2mM EDTA (sterile), 0.5% Bovine Serum Albumin) and incubated with Microbeads conjugated to monoclonal antihuman/mouse CD11b (Mac-1α) antibodies (isotype: rat IgG2b, Miltenyi Biotech). Cd11+ fractions were isolated using MACS LS columns according to manufacturer instructions (Miltenyi Biotech).
Bone marrow derived Mɸ preparation and treatments
Femur and tibia bones from 10-16 weeks-old C57BL6 mice or pepd WT, HET and KO mice were isolated and cleaned, and 10 mL of RPMI-1640 was flushed through each bone using a syringe. Total bone-marrow cells were passed into a 100 μm cell strainer and counted using Countess II automated cell counter (Thermofisher). Cells were spun (400g, 5 min.), resuspended in BMDM culture medium (RPMI1640 supplemented with 20% of L929-conditioned cell medium, 10% heat-inactivated foetal bovine serum (HI-FBS), and 1% penicillin and streptomycin). Total bone-marrow cells were seeded in 10 cm non-culture treated plates (Falcon) at a density of 5x106 cells per plate per 10 ml of Mφ differentiation medium and cultured for 7 days at 37 °C in 5% CO2. On day 5 of differentiation, medium was removed and replace with 10 ml of fresh BMDM culture medium. On day 7, BMDMs were detached using ice-cold PBS-EDTA 1mM, spun (400xg, 5 min.) and resuspended in fresh BMDM culture medium. Differentiated BMDMs were counted using Countess II automated cell counter and cell concentration adjusted to 5x105 cells/ml. Immediately after, cells were plated for experiments at the following densities: 100μl/well of 96-well plate, 500 μl/well of 24-well plate, 1 ml/well of 12-well plate, 2 ml/well of 6-well plate and 10 ml per 10 cm plate. Cells were incubated for 16-24 h after plating before conducting experiments.
Mφ purity of the culture was routinely tested by the expression of CD11b and F4/80 by flow cytometry. 93-97% of the cells express high levels of CD11b and F4/80 after 7 days of differentiation.
To make L929-conditioned medium, L929 cells (CCL-1, ATCC) were seeded in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin-streptomycin and 2 mM L-glutamine (Sigma) at a density of 250,000 cells per 50 ml of medium per T175 tissue culture flask. Medium was harvested after 1 week of culture, and then 50 mL of fresh DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin-streptomycin and 2 mM L-glutamine was added onto cells and harvested 1 week later. Batches obtained after the first and second weeks of culture were mixed at a 1:1 ratio, aliquoted and stored at -20 °C.
After differentiation, BMDMs were cultured in 12 well-culture plate (5 X 105 cell) for 24h in BMDM medium before 6-24h stimulation with LPS (100ng/mL), DEX (100nM), IL4 (10ng/mL), purified PEPD (250nM), Gly-Pro (10mM) or CBZ-Pro (6mM) and stored -80 C prior RNA extraction or prolidase assay. Erlotinib (5µM) was added to the culture medium 2h before and during the treatment with PEPD. We validated PEPD specific effect by measuring cox2 expression in BMDMs, reported as a PEPD target gene13. In addition, we discarded the potential cytotoxic effects of purified PEPD on BMDMs and its potential endotoxin contamination by boiling the purified protein and tested on BMDMs for cox2 induction (Extended data Figure 7a-c).
iPS and monocyte-derived Mɸ differentiation and proteomic analysis
Human blood for monocyte-derived Mɸ was obtained from NHS Blood and Transplant, UK as described12 and all experiments were performed according to guidelines of the University of Oxford ethics review committee. Undifferentiated human iPS cell line was maintained on a monolayer of mitotically inactivated mouse embryonic feeder (MEF) cells in Advanced Dulbecco’s modified Eagles/F12 medium (DMEM/F12), supplemented with 20% Knockout replacement serum (KSR), 2mM L-Glutamine, β-mercaptoethanol (0.055 mM) and 8 ng/ml recombinant human FGF2 (RnD system); and differentiated into MΦ as described previously17. Briefly, this protocol involves key stages of differentiation- i) formation of 3 germ layer containing embryoid bodies (EBs) from iPSCs on withdrawing FGF, ii) long term production of myeloid precursor cells from EBs in presence of 25ng/ml IL-3 and 50ng/ml M-CSF (both RnD) and iii) terminal differentiation and maturation of myeloid precursors into matured MΦ in the presence of higher concentrations of M-CSF (100ng/ml). Protein preparation and proteomic analysis (PXD001953) were performed as described previously18,19. Methods and dataset from the RNAseq (EGAS00001000563) have been described in detail previously12.
Cell Culture and adipocyte differentiation
Primary adipocytes isolated from GnW of 10 weeks-old pepd WT,HET, KO and 3T3L1 Cells were differentiated into adipocytes (day 9) accordingly to the protocol described by Roberts et al.20.
Prolidase activity was determined optimizing Myara's spectrophotometric procedure which was modified from the Chinard technique21,22 and miniaturised in 96 acid resistant well plates. Briefly, Tissue and Mɸ cell extracts were mixed V/V with PBS 50 mM HEPES/1mM MnCl2 and 0.75 mM GSH and incubated 20 min at 50 C. The activated mixture was then added V/V to PBS 94 mM glycyl-proline (Gly-Pro) for a final concentration of 47 mM and incubated or not (control corresponding to basal levels of proline in the cell/tissue extracts) 60 min at 37°C. Reaction was stopped by adding 6V of 0.45 M trichloroacetic acid and centrifuged at 4300 rpm for 60 min. The supernatant (1V) was then added to 4V of a 1:1 mixture of glacial acetic acid and Chinard's reagent (25 g of ninhydrin dissolved at 70 °C in 600 ml of glacial acetic acid and 400 ml of 6 M orthophosphoric acid) and incubated 15-45 min at 90 C. Absorbance was read at 515nm and proline concentration was calculated using proline standards ranged from 0.5 μg to 32 μg. Enzyme activity was reported in micromole of proline released per minute per milligram of protein.
GC-MS analysis of amino acids
Plasma and GnW explants samples were analysed for free amino acid concentrations using the EZ:faast GC-MS Kit (KGO-7166 Phenomenex Inc., Torrance, CA, USA). See Supplementary Information, Supplementary Methods.
AT and liver samples were fixed in 4% paraformaldehyde for 24h, embedded in paraffin, sectioned into 5 μm sections, and processed for Sirius (fibrosis) or haematoxylin and eosin (H&E) (liver steatosis) staining. The slides were scanned (Microscopy Zeiss Axioscan Z1 Slidescanner) and processed for fibrosis (Sirius staining excluding vessels) and steatosis (Vacuole % area) quantification using HALO™ Image Analysis Software.
Hydroxyproline measurement was done using a hydroxyproline colorimetric assay (BioVision) as previously described23. Briefly, frozen fat is weighted and heated in 6 N HCl at 110°C overnight in sealed tubes, as 10 μL of HCl/mg of WAT. Ten microliters are evaporated before incubation with chloramine-T and p-di-methyl-amino-benzaldehyde (DMAB) at 60°C for 90 min. The absorbance was read at 560 nm and the concentration was determined using the standard curve created with hydroxyproline.
Murine and human PEPD protein concentration were measured using respectively an ELISA kit for Mouse Xaa-Pro dipeptidase (PEPD) ELISA kit (CSB-EL017784MO, CUSABIO) and Human PEPD (Peptidase D) ELISA Kit (E-EL-H5575.96, Elabscience) in AT explant (from which debris was removed by centrifugation) and serum according to the manufacturer’s instructions. A standard curve was prepared according to the manufacturer’s instructions, and the value associated with an unconditioned media blank was subtracted from that of conditioned media.
RNA Extraction and Real-Time PCR
RNA from cells extracted using RNeasy Mini columns (Qiagen) according to the manufacturer’s instructions. RNA was harvested from frozen tissue using RNA-STAT-60TM (AMS Bio), and purified by chloroform extraction and isopropanol precipitation. Reverse transcription was performed using Reverse Transcriptase System (Promega) according to manufacturer’s instructions. Real-time PCR was carried out using TaqMan or Sybr Green reagents using an Abi 7900 real-time PCR machine using default thermal cycler conditions. Primer sequences are described in Table S9. Reactions were run in duplicate checked for reproducibility, and then averaged. A standard curve generated from a pool of all cDNA samples was used for quantification. The expression of genes of interest was normalized using the geometric average of four housekeeping genes (18s, 36b4, βactin, and B2m), and data is expressed as arbitrary units.
Regarding human samples (Cohorts 1, 2, 3), RNA purification, gene expression procedures and analyses were performed, as previously described24,25. Briefly, Total RNA was extracted and purified using RNeasy Lipid Tissue Mini kit and integrity was checked by Agilent Bioanalyzer. Total RNA was quantified by means of a spectrophotometer. The same amount of total RNA was reverse transcribed to cDNA from all samples using High Capacity cDNA Archive kit following manufacturers’ instructions. Gene expression was assessed by real-time PCR using a LightCycler 480 real-time PCR system, using TaqMan and SYBRgreen technology suitable for relative genetic expression quantification. The commercially available and pre-validated TaqMan primer/probe sets used are described in Supplementary Information, supplementary Table 9 and 10).
Library preparation and sequencing
Total RNA of GnW was extracted using the miRNeasy mini kit (Quiagen) according to manufacturer’s instructions. Per experiment, 4-10 independent biological repeats were used.
Total RNA was quality checked (RIN >7) via the Agilent Bioanalyser 2100 system, using the Agilent RNA 6000 Nano Kit. 1ug of RNA was used to construct barcoded sequencing libraries with the TruSeq Stranded mRNA HT Sample Prep Kit (Illumina) following supplier’s instruction. All the libraries were validated using the Agilent Bioanalyser DNA 12000 and then multiplexed and sequenced on two lanes of Illumina HiSeq 4000 at SE50 at CR-UK Cambridge Institute Genomics Core Facility.
Processing of RNA-Seq data
RNA-Seq reads (Supplementary Information, supplementary Table 1) were mapped to the most recent ENSEMBLE version GRCm38.p5 of the mouse reference genome sequence (GRCm38.p5) using STAR v2.5.1b26 including the annotations as hints for exon-intron borders. Reads were considered as mapped, if the similarity was at least 95% over at least 90% of the read length as previously described27. FeatureCounts v1.528 was applied for the generation of count tables based on the mapping files. Customized python scripts27 were deployed for downstream processing including the normalization of the raw counts to the total number of assigned reads per gene (TPMs) and to the combined exon length (FPKMs), respectively.
Gene expression and pathway enrichment analyses
Raw counts were subjected to differential gene expression analysis via DESeq229 and different R packages (Supplementary software). Genes, which showed raw counts lower of equal to 2 in 50 % of all samples, were removed prior to the differentially expressed analysis. Wald test was applied to extract differentially expressed genes (DEGs, Supplementary Information, supplementary Table 2, 3, 6-8). Obtained DEGs were annotated via customized python scripts. Pathway enrichment analyses were performed with PIANO30, using the gene set collection C2 retrieved from the Molecular Signatures Database (MSigDB)31,32 (Supplementary Information, supplementary Table 4, 5). Bonferroni and Holm method was applied to correct for multiple testing.
Proteins were extracted from tissue in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EDTA, 1% Triton X-100, pH 7.5) with added protease inhibitor (Roche) and phosphatase inhibitor (Roche) cocktails. Debris and fat were cleared from lysates by centrifugation. Protein concentration was determined by Dc Protein assay (Bio- Rad). After dilution in Laemmli buffer with 0.5% 2-mercaptoethanol, 30 μg protein was loaded per well and subjected to SDS-PAGE in a 4%–12% gradient gel using the Novex NuPage midi system (Life Technologies) and transferred using the iBlot transfer system and reagents (Life Technologies). Membranes were blocked for 1 hour in 3% BSA in tris-buffered saline at room temperature, and incubated overnight at 4oC with the appropriate primary antibody (see Supplementary Information, supplementary Table 9). Bound primary antibodies were detected using peroxidase-coupled secondary antibodies and enhanced chemiluminescence (Millipore). Blots were exposed digitally using the ChemiDoc MP System (Bio-Rad), and bands were quantified using Image J software. The expression of proteins was normalized to protein levels of a housekeeping protein (β-actin or Tubulin), and data is expressed as arbitrary units.
Array based detection of phosphorylated receptor tyrosine kinase
The Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, USA, Catalog Number: ARY014) was employed to screen for the level of phosphorylation of receptor tyrosine kinase (RTKs) in BMDMs in response to purified PEPD, according to the manufacturer’s instructions. Briefly, the array membranes were incubated 1h with an array blocking buffer prior incubation over night at 4C on an orbital shaker with 1.5 ml of cellular extract. The membrane were then washed and incubated 30 min. with streptavidin-HRP solution. Membranes were exposed digitally using the ChemiDoc MP System (Bio-Rad), and spots were quantified using the Image J software. One condition corresponds to a pool of cellular extracts from 4 independent experiments. All the arrays were measured three times; each spots were normalized to the positives controls. The results are presented in a heat map and considered relevant when the fold variation was >1.3 or <0.6.
To determine the cytotoxic effect of compounds on Mɸ, cells were seeded in Roswell Park Memorial Institute (RPMI) 1640 Medium without FBS supplemented at a density of 15,000 cells per well in wells of a 96 well plate. Cells were treated with the given compounds at the given concentrations for 24 hours, and cytotoxicity was measured using an LDH-Cytotoxicity Calorimetric Assay Kit (BioVision) according to the manufacturer’s instructions.
All data from experiments is summarised by its mean, with error bars showing standard error of the mean. The number of replicates is reported in the figure legends. When the results of a pairwise comparison is expressed as a fold-change it is declared in the figure legends to what value the data was normalised. Statistical analysis was performed using Prism7 and Prism8 (GraphPad). Comparisons between two groups were conducted using an unpaired t-test. Comparison between more than two groups were conducted using a one-Way ANOVA followed by appropriate post hoc multiple comparisons tests. Comparisons between more than two groups and factors were conducted using a two-way ANOVA followed by appropriate post hoc testing. Multiple comparisons were corrected and the resulting p-values were adjusted (q-value) relying on the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Pearson’s coefficients were evaluated to estimate the extent of correlation between series of data. Data points were excluded ifrom the correlation analysis f they exhibited a value of more than two SDs from the mean. For all metabolic tests, the model animals were randomly assigned to the different experimental settings (GTT, ITT). Areas under the receiver operating characteristic (ROC) curves were determined for each variable to identify the predictors of AT fibro-inflammation and insulin resistance/type 2 diabetes. ROC curves is a plot of sensitivity (true positive) versus 1–specificity (false positive) showing the ability of biomarker (PEPD level) to discriminate between true positives (e.g. insulin resistant) and true negatives (e.g. insulin sensitive). The best marker has an ROC curve shifted to the left with area under the curve close to unity33. To determine the optimal cut-off values for fibro-inflammatory status or insulin resistance indices, the Youden index was calculated (sensitivity + specificity−1), and the values for the maximum of the Youden index was considered as the optimal cut-off points using the Web-tool easyROC34. Statistical significance was set at *p <0.05, **p<0.01 and ***p<0.001.
Exploratory Factor Analysis
Exploratory factor analysis (EFA) was conducted to determine the possible latent structure of the variables (listed, e.g., in Figure 1.p – rows of the depicted matrix) measured in each animal models, i.e., pepd mice, CBZ-Pro-treated mice and BMT mice. Factor analysis identifies a minimum number of new variables (factors) which are linear combinations of the original (measured) ones such that the new (fewer) variables contain most or all of the information and can facilitate the interpretation of a complex multivariate scenario. First, a matrix of correlation coefficients is computed and, from it, a set of main components (factors) is extracted. The relationships between the original variables and the factors is expressed in terms of “factor loadings” which, broadly speaking, can be interpreted as an estimate of the degree of correlation between the original variables and the factors.
We determined whether our data were appropriate for EFA by using the Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity (REF). Data were considered appropriate if the KMO was >0.7 and Bartlett’s test was significant at P < 0.05. To determine the number of factors to retain we decided to rule out the components associated with eigenvalues less than 1. Parallel analysis confirmed that our choice was viable. Factors extraction was computed using the Maximum Likelyhood Estimate (MLE) method, implemented in the function fa of the psych R package35. Plots were produced either using in house scripts or using built-in functions of the FactoMineR R package36.
Principal components analysis
Principal components analysis of the Lukk and the own dataset were calculated in R version 3.1.2 using the prcomp function of the stats package37.