Journal of Crohn's and Colitis, 2022, 16, 1571–1583 https://doi.org/10.1093/ecco-jcc/jjac069 Advance access publication 12 May 2022 Original Article The Gut Microbiota Metabolite Succinate Promotes Adipose Tissue Browning in Crohn’s Disease Diandra Monfort-Ferré,a,b Aleidis Caro,c Margarita Menacho,d Marc Martí,e Beatriz Espina,c Albert Boronat-Toscano,a,b Cati Nuñez-Roa,a,b Jesús Seco,a,b Michelle Bautista,d Eloy Espín,e Ana Megía,a,b Joan Vendrell,a,b,f Sonia Fernández-Veledo,a,b, Carolina Serenaa,b, a Hospital Universitari de Tarragona Joan XXIII, Institut d’Investigació Sanitària Pere Virgili, Tarragona, Spain b CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain c Colorectal Surgery Unit, Hospital Universitari Joan XXIII, 43007, Tarragona, Spain d Digestive Unit, Hospital Universitari Joan XXIII, 43007, Tarragona, Spain e Colorectal Surgery Unit, General Surgery Service, Hospital Valle de Hebron, Universitat Autonoma de Barcelona, 08035, Barcelona, Spain f Universitat Rovira i Virgili, Tarragona, Spain Corresponding authors: Sonia Fernández-Veledo, PhD, Hospital Universitari de Tarragona Joan XXIII, Institut d’Investigació Sanitària Pere Virgili, Tarragona, Spain. Email: sonia.fernandezveledo@gmail.com; sonia.fernandez@iispv.cat; and Carolina Serena, PhD, Hospital Universitari de Tarragona Joan XXIII, Institut d’Investigació Sanitària Pere Virgili, Tarragona, Spain. Email: carolserena@gmail.com; carolina.serena@iispv.cat Abstract Background and Aims: Crohn’s disease [CD] is associated with complex microbe–host interactions, involving changes in microbial communi- ties, and gut barrier defects, leading to the translocation of microorganisms to surrounding adipose tissue [AT]. We evaluated the presence of beige AT depots in CD and questioned whether succinate and/or bacterial translocation promotes white-to-beige transition in adipocytes. Methods: Visceral [VAT] and subcutaneous [SAT] AT biopsies, serum and plasma were obtained from patients with active [n = 21] or inactive [n = 12] CD, and from healthy controls [n = 15]. Adipose-derived stem cells [ASCs] and AT macrophages [ATMs] were isolated from VAT biopsies. Results: Plasma succinate levels were significantly higher in patients with active CD than in controls and were intermediate in those with inactive disease. Plasma succinate correlated with the inflammatory marker high-sensitivity C-reactive protein. Expression of the succinate re- ceptor SUCNR1 was higher in VAT, ASCs and ATMs from the active CD group than from the inactive or control groups. Succinate treatment of ASCs elevated the expression of several beige AT markers from controls and from patients with inactive disease, including uncoupling protein-1 [UCP1]. Notably, beige AT markers were prominent in ASCs from patients with active CD. Secretome profiling revealed that ASCs from patients with active disease secrete beige AT-related proteins, and co-culture assays showed that bacteria also trigger the white-to-beige switch of ASCs from patients with CD. Finally, AT depots from patients with CD exhibited a conversion from white to beige AT together with high UCP1 expres- sion, which was corroborated by in situ thermal imaging analysis. Conclusions: Succinate and bacteria trigger white-to-beige AT transition in CD. Understanding the role of beige AT in CD might aid in the devel- opment of therapeutic or diagnostic interventions. Key Words: Succinic acid; SUCNR1; beige adipose tissue; bacteria translocation, creeping fat Graphical Abstract Adipose tissue creeping fat Crohn’s disease Succinate accumulation Beiging Bacterial translocation UCP-1-gene and -protein expression © The Author(s) 2022. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1572 D. Monfort-Ferré et al. 1. Introduction Endoscopic evaluation was performed in 80% of the pa- tients, with complete correspondence with the clinical classi- Crohn’s disease [CD] is chronic disorder of the gastrointes- fication obtained by CDAI.22,23 tinal tract characterized by severe transmural inflammation Samples of subcutaneous [SAT] and visceral [VAT, mesen- with destruction of the intestinal barrier, which increases in- teric origin] AT were obtained from patients with inactive testinal permeability. A recent study1 suggested that bacterial CD [n = 12] and from patients without CD [control group] translocation across the ‘leaky gut’ facilitates access to the [n = 15] undergoing non-acute surgical interventions such mesenteric fat and the development of an inflammatory re- as hernia or cholecystectomy, in a scheduled routine surgery. sponse in the adipose tissue [AT] surrounding the damaged Samples of mesenteric perilesional [81% ileum, 5% colon and intestinal area, termed creeping fat. 14% ileocolon] ‘wrapping’ VAT [creeping fat origin] and SAT Microbial dysbiosis in CD has been associated with an [n = 21] were obtained from patients with active CD sched- increase in succinate-producing bacteria and a decrease in uled for surgery for symptomatic complications or failure of succinate-consuming bacteria,2 which is in good agreement medical therapy. All tissue samples were aseptically collected. with the elevated succinate levels reported in the intestine Clinical data, and anthropometric, demographic and bio- and faeces of patients with this condition.3–5 Classically con- chemical variables of the cohort are shown in Table 1. Groups sidered as a marker of hypoxia and tissue damage, succinate were matched by body mass index [BMI], gender and age. is now recognized as a pro-inflammatory signal of immune activation.6–9 Recent observations support additional func- 2.2. UCP1 immunohistochemistry tions of succinate; for example, we and others have dem- Tissues were fixed in 10% neutral formalin buffer overnight, onstrated that succinate serves a key role in fine-tuning the embedded in paraffin, sectioned and mounted on glass slides. inflammatory response by acting both as an alarmin and a The anti-UCP1 polyclonal rabbit antibody [ab10983; recom- resolving molecule.10–13 Furthermore, it was reported that mended dilution 1:500] used was purchased from Abcam. succinate oxidation drives AT thermogenic respiration via the production of reactive oxygen species14; the same au- 2.3. Isolation and incubation of AT explants thors later described that brown adipocytes [specialized AT cells that dissipate energy as heat] control liver extracellular VAT samples were obtained from the control group [n = 4] succinate levels in an obesogenic environment regulated and from the active [n = 4] and inactive [n = 4] CD groups. by the inner mitochondrial membrane protein uncoupling Two fresh explants of each mesenteric VAT biopsy [~0.2 g] protein-1 [UCP1].15 Succinate has also been described as an were placed in 1 mL of DMEM/F12 medium containing inductor of brown-like adipocyte progenitors within white 10% fetal bovine serum [FBS]. Samples were incubated adipose depots [so-called brown-in-white, brite or beige adi- without or with succinate [250 μM] for 24 h at 37°C and pocyte progenitors].16 In line with this latter potential func- 5% CO2. tion of succinate, here we hypothesized that the persistent 2.4. AT-derived stem cells elevated levels of succinate in areas of intestinal damage in CD would trigger browning of the mesenteric creeping fat ASCs were isolated as described.24,25 Briefly, VAT biopsies by inducing the beige adipogenesis programme in adipose- were washed extensively with PBS to remove debris and were derived stem cells [ASCs] as a compensatory mechanism treated with 0.1% collagenase in PBS–1% bovine serum al- to ameliorate inflammation and fibrosis. We further hy- bumin [BSA] for 1 h at 37°C with gentle agitation. Digested pothesized that the presence of bacteria in the creeping fat samples were centrifuged at 300 g at 4°C for 5 min to sep- would trigger metabolic changes in this tissue and induce arate adipocytes from stromal cells. The cell pellet containing browning. There is some evidence for this in the literature. the stromal fraction was re-suspended in stromal culture me- Bacterial infections have been associated with an increase in dium consisting of DMEM/F12, 10% FBS and 1% antibiotic/ pathogen-specific memory T cells,17 which respond to bac- antimycotic solution. To prevent spontaneous differentiation, teria by secreting antimicrobial peptides18 and recruiting primary ASC cultures at passage 0 [P0] were grown to 90% beige adipocytes19 in white AT. Furthermore, other infections confluence and harvested with trypsin-EDTA, and aliquots such those produced by viruses promote beige adipogenesis [1 × 106 cells] were cryopreserved in liquid nitrogen until re- in preadipocytes.20,21 quired.26 Adipose tissue [VAT]-derived macrophages [ATMs] were also isolated from the stromal vascular fraction of AT biopsies as described.24,27–29 2. Material and Methods 2.5. Adipose stem cell immunophenotyping 2.1. Study design Cells [2 × 105] were incubated with a panel of primary anti- Subjects were recruited at University Hospital Joan XXIII bodies [described in Supplementary Table 1].30 After iso- [Tarragona, Spain] and University Hospital Vall d’Hebrón lation, the minimal functional and quantitative criteria, [Barcelona, Spain] in accordance with the tenets of the established by the International Society of Cell Therapy and Helsinki Declaration. All participants gave their informed the International Federation for Adipose Therapeutics and consent, and the study was reviewed and approved by Science, were confirmed by flow cytometry.24,30 All experi- the ethics and research committees of the respective hos- ments were performed in cells at P3–P5. pitals [references CEIM 177/2018; CEIM 41p/2015; PR(CS)383/2021]. Donors were classified as those in re- 2.6. RNA extraction lapse [active] or in remission [inactive] following the Crohn’s RNA was extracted from 200 mg of AT or AT explants, or Disease Activity Index [CDAI] criteria and also based on from ASCs [100 000 cells per well] using the TriPure Isolation biological and clinical parameters such as high-sensitivity Reagent [Roche]. RNA concentration was determined by C-reactive protein [hsCRP] and faecal calprotectin levels. absorbance at 260 nm, and purity was estimated with a Adipose Tissue Browning in Crohn’s Disease 1573 Table 1. Demographic characteristics and clinical data Control Active CD Inactive CD N 15 21 12 Sex [male/female] 10/5 10/11 5/7 Age 50.8 ± 9.1 40.3 ± 12.28 46.4 ± 14.69 BMI [kg/m ]2 24.51 ± 1.9 24.49 ± 3.8 24.80 ± 7.4 Glucose [mg/dL] 81 ± 3.5 78.81 ± 17.58 84 ± 11.29 Cholesterol [mg/dL] 115 ± 15 138.4 ± 30.4 122.5 ± 17.36 HDLc [mg/dL] 38.6 ± 3.5 43.94 ± 14.45 31.13 ± 8.4 Triglycerides [mg/dL] 97.4 ± 5.9 102 ± 54.63 147.54 ± 96.6a Smoking status, n [%] Current-smoker 5 [3.3] 5 [23.8] 2 [16.6] Never-smoker 6 [40] 14 [66] 6 [50] Ex-smoker 4 [26.6] 2 [9.5] 4 [9.5] Age at diagnosis, n [%] A1 — 1 [4.8] 4 [33.3] A2 — 17 [80.9] 3 [25] A3 — 3 [14.3] 5 [41.6] Location, n [%] L1 — 17 [80.9] 8 [66.6] L2 — 1 [4.8] 2 [16.6] L3 — 3 [14.3] 2 [16.6] Behaviour, n [%] B1 — 2 [9.5] 5 [41.6] B2 — 9 [42.8] 4 [33.3] B3 — 10 [47.6] 3 [25] Corticoid treatment, n [%] — 9 [52.9] 6 [50] Biological treatment, n [%] — 10 [58.8] 7 [58.3] C-reactive protein [mg/dl] 0.15 ± 0.06 3.12 ± 0.98a 0.77 ± 0.16a,b Faecal calprotectin [μg/g] — 2219 ± 290.4 369.3 ± 216.5b Succinate [μM] 42.46 ± 5.06 128.82 ± 20.9 a 88.82 ± 10.1a,b Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; age at diagnosis: A1 ≤ 16 years; A2 17–40 years; A3 > 40 years; location: L1 = ileal; L2 = colonic; L3 = ileocolonic; behaviour: B1 = no stenotic, no fistulizing Crohn’s disease; B2 = stenotic Crohn’s disease; B3 = fistulizing Crohn’s disease. a p <0.05, significant differences compared with healthy control; b p <0.05, significant differences compared with patients with active disease. Nanodrop spectrophotometer [Nanodrop Technologies]. 2.9. Western blotting cDNA was synthesized using SuperScript II reverse tran- SAT and VAT were lysed and homogenized in M-PER buffer scriptase and random hexamer primers [Invitrogen Life containing a Protease Inhibitor Cocktail, and protein con- Technologies]. centration was determined with the BCA Protein Assay Kit [all from Pierce Biotechnology]. Equal amounts of total protein were separated by SDS-polyacrylamide gel electro- 2.7. Real-time quantitative PCR phoresis, transferred to Immobilon membranes [Millipore] Quantitative gene expression was evaluated by real-time PCR and blocked. Membranes were probed with polyclonal anti- [qPCR] on a 7900HT Fast Real-Time PCR System using the bodies against UCP1 [Abcam], PRDM16 [ThermoFisher TaqManR Gene Expression Assay [Applied Biosystems]. Scientific] and β actin [Sigma-Aldrich], which was used as Genes are listed in Supplementary Table 2]. a loading control. Immunoreactive bands were visualized with SuperSignal West Femto chemiluminescent substrate [Pierce Biotechnology] and images were captured using 2.8. UCP1 immunofluorescence in ASCs the VersaDoc imaging system and Quantity One software ASCs isolated from patients with CD were seeded at 20 000 [BioRad]. cells/cm2 and co-cultured or without bacteria and treated with succinate [250 μM] or left untreated. Cells were fixed with 4% paraformaldehyde and then permeabilized with 2.10. Serum treatment of ASCs 0.25% Triton X-100. Cells were then stained with the anti- ASCs isolated from healthy controls were seeded at 20 000 UCP1 polyclonal rabbit antibody [dilution 1:200]. PBS was cells/cm2, and serum from controls and from patients with in- used as a negative control. active or active CD were added to cells at a 1:10 ratio [serum/ 1574 D. Monfort-Ferré et al. culture medium]. Cells were collected 3 h later to assess UCP1 control wells, which received 500 µL of DMEM/F12 medium. gene expression. ASCs were incubated with bacteria at 37°C for 1, 3 and 6 h [Supplementary Figure 1A]. Based on preliminary results, an 2.11. Proteomics analysis of conditioned medium incubation time of 3 h was chosen to perform the subsequent from ASCs experiments. For control-ASCs, a 1-h pre-stimulus with lipo- We used an untargeted proteomics approach consisting of li- polysaccharide [LPS] at [100 ng/mL; Sigma-Aldrich] was used quid chromatography coupled to tandem mass spectrometry to mimic a pro-inflammatory condition. ASCs were then col- to compare and identify differences in proteins secreted into lected and used for gene expression analysis. the extracellular [conditioned] medium by ASCs isolated from the creeping fat of patients with active CD [n = 9], ASCs iso- 2.13. Succinate determination lated from the VAT of patients with inactive CD [n = 8] and Circulating plasma succinate levels and extracellular succinate ASCs isolated from the VAT of healthy individuals [n = 9]. from conditioned media of AT explants were measured with the EnzyChromTM SuccinatecAssay Kit [BioAssay Systems], 2.11.1. Sample preparation as described.10,24,31,32 The assay sensitivity was 12 μM and the ASCs were cultured at 94 000 cells/cm2 with 0.9 mL of col- intra- and interassay coefficients of variance were less than ourless culture medium [supplemented with 2% BSA and 3.5% and 6.95%, respectively. 1% antibiotic/antimycotic solution without FBS]. After 9 h, the conditioned medium was collected and stored at −80°C. 2.14. Bio-impedance and infrared thermography Before analysis, 400 µL of secretome samples were concen- Infrared thermography and bio-impedance were performed trated to 50 µL in AmiconUltra 3 K columns [Millipore]. in 12 healthy subjects, and in 11 subjects with CD [five active and six inactive], at the Hospital Universitari Joan XXIII. For 2.11.2. Chromatography and mass spectrometry infrared thermography, environmental, technical and indi- analysis vidual factors were controlled as previously recommended,33 Proteomics analysis was performed at the Centre for Genomic and FLIR Tools was used to analyse the images [https://www. Regulation and University Pompeu Fabra [Barcelona, Spain] flir.com/browse/professional-tools/thermography-software/]. using an LTQ-Orbitrap Fusion Lumos mass spectrometer Bioelectrical impedance analysis was used to estimate body [ThermoFisher Scientific] coupled to an EASY-nLC 1200 in- mass composition, body water volume, total energy con- strument [ThermoFisher Scientific/Proxeon Biosystems] using sumption, bioelectrical phase angle and several indices of fat a 90-min chromatographic gradient. mass.34 2.11.3. Data analysis 2.15. Statistical analysis Acquired spectra were analysed using the Proteome Statistical analysis was performed with the Statistical Package Discoverer software suite [v2.3, ThermoFisher Scientific] for the Social Sciences software, v15 [SPSS]. For clinical and and the Mascot search engine [v2.6, Matrix Science], as de- anthropometrical variables, normally distributed data were scribed.10 Proteomic data were evaluated to identify signifi- expressed as mean ± SD; for variables with no Gaussian dis- cant differences [p < 0.05] in proteins between the different tribution, values were expressed as median [25th–75th quar- groups. Proteins that were not present in six of the nine sam- tiles]. Student’s t-test with Bonferroni adjustment was used to ples were not considered for statistical analysis. All statistical compare the mean value of normally distributed continuous data treatments were performed with the R statistical pro- variables. For variables that did not have a Gaussian distri- gramming environment [v3.5.3; R Core Team, 2019]. The bution, we used the Kruskal–Wallis test with post hoc Dunn’s mass spectrometry proteomics data have been deposited in multiple comparisons test. To analyse the differences in nom- ProteomeXchange with accession code PXD030520. For inal variables between groups, we used the χ2 test. Pearson’s functional studies, STRING v11 protein–protein association correlation coefficient with Bonferroni adjustment was used networks [https://string-db.org/] were used to study the impli- to analyse the relationship between parameters. For in vitro cated molecular and functional pathways for the significant data, experimental results are presented as mean ± SEM. differentially secreted proteins. Comparisons among the three groups were performed using the non-parametric Krustal–Wallis test. Visualizations were 2.12. Human ASCs co-cultured with Escherichia performed with GraphPad Prism v6 [Graphpad Software]. coli as an infection model Escherichia coli [strain ATCC 25922 and an independent 3. Results clinical strain] and a commensal strain of Lactobacillus spp. were kindly provided by Dr Terron-Puig from the Hospital 3.1. Circulating levels of succinate correlate laboratory of Joan XXIII of Tarragona. All three strains were positively with SUCNR1 expression in AT of CD cultured on agar plates and colonies were incubated at 37°C patients for 24–72 h. In parallel, ASCs at passage 3–5 were seeded Plasma succinate levels were significantly higher in patients at 60 000 cells per well in 12-well culture plates. Bacterial with active CD than in healthy controls [Figure 1A], in line colonies were picked and resuspended in 2 mL PBS and the with previous observations,5,35 whereas patients with inactive bacterial inoculum was determined by McFarland turbidity disease showed intermediate levels [Figure 1A]. Circulating standards and counted in a Neubauer chamber. Briefly, 1 × 107 succinate levels correlated positively with the levels of the CFU/mL of bacterial suspension was centrifuged, washed pro-inflammatory marker hsCRP [Figure 1B]. and resuspended in DMEM/F12 medium without antibiotic/ Extracellular succinate governs local stress and inflam- antimycotic solution. Subsequently, 500 µL of bacterial sus- matory responses by binding its cognate receptor SUCNR1/ pensions were added to each well with the exception of the GPR91,8,36 the expression of which is elevated in the affected Adipose Tissue Browning in Crohn’s Disease 1575 mucosa of patients with CD,35 promoting pro-inflammatory increase in UCP1, PRDM16, CPT1A, CPT1B, ELOVL3 and and pro-fibrotic processes. Gene expression analysis of SAT ADRB3 gene expression [Figure 2A]. The expression level of samples revealed no significant changes in SUCNR1 levels be- several of these genes was intermediate in ASCs from patients tween healthy controls and patients with or without active with inactive disease except for TBX1, which was signifi- CD [Figure 1C]. By contrast, expression analysis of samples cantly higher in the inactive group than in the control and of the surrounding VAT, in close contact with the bowel, re- active groups [Figure 2A]. vealed significantly higher SUCNR1 gene expression in the We next used secretome profiling to survey the proteins se- VAT [creeping fat origin] from patients with active disease creted by ASCs isolated from patients with CD [active and than from controls, and significantly lower expression in sam- inactive] and from healthy controls [Figure 2B]. Network ples from patients with inactive disease [Figure 1D]. Similar analysis of proteins secreted more in the conditioned medium results were found for ATMs and ASCs isolated from the VAT of ASCs from patients revealed a significant protein–protein depots of the three groups [Figure 1E and F, respectively], and interaction enrichment of the network [p < 1.0 × 10−16] SUCNR1 expression in VAT-derived ASCs correlated posi- [Supplementary Figure 2]. We found a significant increase in tively with circulating plasma levels of succinate [Figure 1G]. proteins of several pathways including extracellular matrix Given that SUCNR1 engagement in AT is likely to modulate components [POSTN, VCAN, COL12A1, LOXL2, and its inflammatory status,8,10,36 we treated VAT explants with FBN1], and cell differentiation [Figure 2C], with several pro- succinate and surveyed the gene expression of several pro- teins related to browning including VCL, TIMP1, LTBP2, inflammatory cytokines. Explants from patients with active IGFBP3, IGFBP5 and FSTL1.37–40 The last three proteins disease showed a consistent increase in cytokine gene expres- [IGFBP3, IGFBP5, FSTL1] were lower in abundance in pa- sion after the addition of succinate when compared with the tients with inactive CD.41,42 other groups [Figure 1H]. Of note, the addition of succinate induced an ‘anti-inflammatory’ gene response in VAT samples 3.4. Bacterial colonization triggers browning in of healthy controls, which was mimicked in VAT samples of ASCs from patients with CD patients with inactive disease [Figure 1H], similar to what has Bacterial translocation from the gut to the AT is a well- been reported in the SAT of healthy subjects.9 Succinate levels recognized phenomenon in CD, prompting the growth of were significantly higher in the conditioned medium of AT creeping mesenteric fat that envelops the affected intestinal explants isolated from patients with active CD than from pa- wall and activates AT immune cells.1 We recently showed that tients with inactive CD and from control subjects [Figure 1I]. ASCs from patients with CD have unique immune proper- ties including phagocytosis, which can be activated by bac- 3.2. Adipocyte precursors from patients with CD terial signals.24 We performed co-culture experiments of ASCs adopt a brown-like phenotype with a clinical E. coli strain to investigate whether bacteria Because succinate is recognized as an inducer of beige could switch on a browning gene programme. As shown in adipogenesis in white adipose tissue progenitors,16 we ques- Figure 3A, the presence of E. coli triggered a marked increase tioned whether it could influence the characteristics of ASCs in TNFA expression in all groups of ASCs, indicating acti- in creeping fat. We thus treated ASCs isolated from the mes- vation of the inflammatory pathway [probably to resolve enteric VAT of control and patients with CD [active and the bacterial infection]. By contrast, UCP1 gene expression inactive] ex vivo with succinate and evaluated UCP1 gene was triggered only in ASCs from patients with CD [Figure expression. We noted an increase in the expression of UCP1 3A middle and lower panel]. This observation suggests that, in samples from controls and from patients with inactive in healthy conditions, a bacterial stimulus is not sufficient to disease after the addition of succinate to the medium [Figure trigger browning in adipocyte precursors. Furthermore, E. 1J]. These findings indicate that succinate probably induces coli co-culture with ASCs from patients with active CD trig- browning adipogenesis in precursors both from healthy con- gered the expression of RAB5A [Figure 3A middle panel], a trols, as previously described,16 and also from patients with regulator of the phagocytosis pathway.24,43 We repeated the inactive CD. By contrast, succinate failed to induce UCP1 experiments using a reference strain of E. coli [ATCC 25922] gene expression in samples from the active disease group, and also a commensal strain of Lactobacillus spp. Of note, which showed already high basal levels of UCP1 [Figure 1J], both strains were able to increase TNFA gene expression but probably due to exposure to an environment of elevated suc- only the E. coli reference strain was able to significantly in- cinate in vivo. Supporting this idea, we found that the incuba- crease UCP1 and RAB5A gene expression as compared with tion of ASCs from healthy subjects with serum from patients cells without the addition of bacteria. These findings indicate with active CD [in whom succinate levels are higher] signifi- that E. coli strains can stimulate the beiging programme in cantly increased UCP1 expression [Figure 1K], and we found VAT [creeping fat] of CD patients. To test the influence of a a positive relationship between UCP1 expression in ASCs and pro-inflammatory environment on ASCs, we used LPS to pre- circulating succinate levels [Figure 1L]. These findings sug- stimulate cells from control subjects before their co-culture gest that ASCs isolated from patients with active CD display with E. coli. Results showed that an acute inflammatory a brown-like phenotype with high expression of UCP1, and stimulus was not sufficient to increase UCP1 gene expression high UCP1 gene expression is linked to high succinate levels in control ASCs [Supplementary Figure 1C, left panel]. We re- in patients with active CD. peated the experiment in ASCs from patients with active CD, finding that an acute inflammatory stimulus plus the addition 3.3. The brown-like phenotype of ASCs from of bacteria did not further increase the gene expression of patients with CD depends on the disease activity UCP1 compared with bacteria alone [Supplementary Figure To validate the browning phenotype of ASCs isolated from 1C, right panel]. Immunofluorescence staining of UCP1 in patients with active CD, we first tested the expression of other ASCs from patients with active CD [pre-treated or with bac- beige/brown adipocyte marker genes, finding a significant teria or untreated] confirmed the gene expression findings and 1576 D. Monfort-Ferré et al. A $ B C Control R2 = 0.641 2.0 200 * 400 Active CD P = 0.0004 SAT SUCNR1 mRNA Circulating succinate 1.5 150 Inactive CD 300 Hs CRP (mg/L) levels (µmolar) 100 * 200 1.0 100 50 0.5 0 0 0 5 10 15 0.0 Circulating succinate (µmolar) D E F G $ R2 = 0.438 2.5 $ 4 $ 80 800 P < 0.0001 * * ASC SUCNR1 mRNA ATM SUCNR1 mRNA VAT SUCNR1 mRNA ASC SUCNR1 mRNA 2.0 3 * 60 600 1.5 2 400 40 1.0 * 200 1 20 0.5 0 0.0 0 0 100 200 300 0 Circulating succinate (µmolar) H 2.0 1.5 1.5 1.5 I 1.5 Adipose tissue CCL2 mRNA TNFA mRNA IL1B mRNA 1.0 1.0 IL6 mRNA 1.0 explants Control 1.0 * 400 * 0.5 0.5 0.5 Succinate levels conditioned 0.5 * * 300 0.0 media (µmolar) 0.0 0.0 0.0 Succinate – + – + – + – + Adipose tissue explants (250 µM) 200 3 * 4 2.5 4 * * 2.0 * Active CD CCL2 mRNA 3 3 TNFA mRNA 100 L1BI mRNA IL6 mRNA 2 1.5 2 2 1.0 0 1 1 1 0.5 0 0 0.0 0 Succinate – + – + – + – + (250 µM) 1.5 1.5 2.5 2.0 2.0 TNFA mRNA Inactive CD 1.5 CCL2 mRNA 1.0 IL1B mRNA IL6 mRNA 1.0 1.5 1.0 * 1.0 0.5 * 0.5 * 0.5 0.5 0.0 0.0 0.0 0.0 Succinate – + – + – + – + (250 µM) J K 3 Control- ASC Serum: L 80 * * Control 500 60 R2 = 0.85 Active CD UCP1 gene expression UCP1 gene expression 400 P < 0.0001 40 * Inactive CD Serum succinate $ 2 20 300 (µmolar) * 8 200 6 1 100 4 * 2 0 0 0 0.5 1.0 1.5 2.0 2.5 Succinate – + – + – + UCP1 gene expression (250 µM) Control-ASC CDa-ASC CDi-ASC Figure 1. Succinate levels are elevated in the active phase of Crohn’s disease and correlate positively with UCP1 gene expression in pre-adipocytes. [A] Circulating succinate plasma levels in controls [healthy subjects] [n = 10] and in patients with active [n = 17] or inactive [n = 12] Crohn’s disease [CD]. Differences were analysed with the Kruskal–Wallis test with post hoc Dunn’s multiple comparison test. *p < 0.0001 vs. control; $p < 0.05 vs. active CD. [B] Positive correlation between plasma succinate and high-sensitivity C-reactive protein [hsCRP] levels. [C] SUCNR1 expression in subcutaneous [SAT] and [D] visceral [VAT] adipose tissue [n = 8 per group], and in [E] VAT-derived adipose tissue macrophages [ATMs] [n = 5 per group] and [F] VAT-derived stem cells [ASCs] [n = 8 per group]. [G] Positive correlation between visceral-ASC SUCNR1 mRNA and circulating succinate. [H] Succinate treatment differentially affects adipose tissue of patients with CD according to clinical status. Quantitative PCR analysis of pro-inflammatory genes in VAT explants, unstimulated or stimulated with succinate [250 µM] for 24 h, from controls and from patients with active or inactive CD [n = 4 per group]. [I] Succinate release in 24-h-conditioned medium of VAT explants from controls and from patients with active or inactive CD [n = 4 per group]. [J] UCP1 expression in ASCs from controls and from patients with active or inactive CD after succinate treatment. [K] Serum from patients with active CD [which has a high amount of succinate] increases UCP1 gene expression in ASCs isolated from controls. [L] Positive correlation between UCP1 expression in ASCs and plasma succinate levels. Pearson’s correlation coefficient with Bonferroni adjustment was used to analyse the relationship between parameters. *p < 0.05 vs. control; $p < 0.05 as indicated in the figure. Adipose Tissue Browning in Crohn’s Disease 1577 A ASCs-Control 25 $$ ASCs-Active CD ASCs-Inactive CD 20 * Relative gene expression 15 $ $$ 10 * $ ** $$ $ 5 * ** * 0 UCP1 PRDM16 TMEM26 CPT1A CPT1B ELOVL3 TBX1 ADRB3 COX7A1 FOXC2 PPARGC1 PPARGC1B B ASC proteome Elevated in control Elevated in crohn VCAN POSTN Extracellular matrix organization (FDR = 1.5e-18) 8 Cell differentiation (FDR = 7.9e-07) VCL COL12A1 6 LOXL2 IGFBP3 –log10 (p) TPM1 4 FSTL1 LTBP2 FBN1 IGFBP5 2 –4 –3 –2 –1 0 1 2 3 4 Log (FC) C 1. Extracellular matrix components POSTN VCAN COL12A1 LOXL2 TIMP2 CDH13 $ ASCs-Control 9 8 × 10 2.5 × 108 ASCs-Active CD Protein abundance * * 4 × 10 8 6 × 10 8 2.0 × 10 9 2.0 × 10 9 * * 6 × 109 2.0 × 108 3 × 108 * 1.5 × 109 1.5 × 109 * ASCs-Inactive CD 1.5 × 10 8 * 4 × 108 4 × 10 9 * 2 × 108 1.0 × 109 * 1.0 × 109 1.0 × 108 2 × 10 9 2 × 108 8 8 8 5.0 × 10 5.0 × 10 7 1 × 10 5.0 × 10 * 0 0.0 0 0.0 0 0.0 2. Cell differentiation (browning-related molecules) FSTL1 PCOLCE VCL IGFBP5 IGFBP7 $ 8 $ 9 1.5 × 109 6 × 109 2.0 × 109 1 × 10 3 × 10 * * Protein abundance * * 7 * 9 8 × 10 1.5 × 10 1.0 × 109 4 × 109 2 × 109 6 × 107 1.0 × 109 * 4 × 107 2 × 109 1 × 109 5.0 × 108 5.0 × 108 2 × 107 0.0 0 0 0.0 0 Figure 2. Adipose stem cells isolated from patients with Crohn’s disease show a brown-like phenotype. [A] Relative gene expression of browning markers in adipose stem cells [ASCs] from visceral adipose tissue [VAT] of patients with active [n = 6] or inactive [n = 6] Crohn’s disease [CD] or from healthy controls [n = 3]. [B, C] Analysis of the ASC protein secretome and protein abundance differences between ASCs of the control group [n = 9] and of the active [n = 9] and inactive [n = 8] CD groups. Shown are examples of enriched pathways [extracellular matrix components and cell differentiation pathways]. Bars in graphs represent mean ± SEM. *p < 0.05 vs control; $p < 0.05 and $$p < 0.01 as indicated in the figure. 1578 D. Monfort-Ferré et al. revealed an enhancement of UCP1 expression after E. coli unknown. It has been proposed that adipocytes from creeping co-culture [Figure 3B], which was corroborated after quanti- fat maintain their ability to counteract inflammation and may fication of the signal [Figure 3C]. possibly serve a protective function.47 In this sense, the white- to-beige transition of adipocytes may explain the increase of 3.5. Beige phenotype of AT in patients with CD is anti-inflammatory cytokines reported in the creeping fat of associated with higher body temperature patients with CD.46,47 Of note, our proteomics data revealed a Finally, we explored whether the browning phenotype evident high abundance of follistatin from CD-ASCs, which is a posi- in adipocyte precursors obtained from the VAT of patients tive regulator of brown adipose tissue function by blocking with CD was also detectable in whole tissue and organs. TGF-β signalling pathways and GDF8 [myostatin] actions, UCP1 expression was significantly higher in AT samples of and by exerting anti-inflammatory effects.41,48,49 Indeed, white patients with CD than of healthy controls, both in VAT and AT precursor cells can be modified with different factors to SAT depots, and with a clear predominance in VAT of patients produce AT that is more energetically active.50,51 Extracellular with active disease [Figure 4A]. We also analysed the protein signals include activation of the sympathetic nervous system, expression of UCP1 and PRDM16 in SAT and VAT depots by immune cells and epigenetic changes,52 which influence the western blotting. Results showed that VAT UCP1 levels were transcription of specific genes.53 Because beige adipocytes significantly greater in patients with active disease [Figure 4B]. utilize glucose, succinate and other metabolites, they essen- In good agreement with this, immunohistochemical analysis tially clear these products from the circulation, which in- [Figure 4C] and quantification [Figure 4D] revealed higher creases glucose and insulin sensitivity.14,54 Accordingly, the UCP1 staining in both the SAT and VAT depots of patients oxidation of local and circulating succinate during browning with active or inactive disease than in healthy controls. probably reduces its levels, in turn reducing AT inflammation When we expanded the analysis to several other genes and fibrosis specifically in creeping fat. An important point of expressed in beige/brown fat cells, many of them were ele- discussion is the origin of succinate as it could be produced vated in the VAT from patients with CD [Figure 4E], and both by the host and the gut microbiota. In this sense, pre- some showed a differential behaviour depending on whether vious studies point to gut microbiota as the main source of the disease was active or inactive [ADRB3 and COX7A1]. succinate in CD.5 However, it should be not ruled out that Intriguingly, we found an increase of the beige cell progenitor AT, the intestine and other damaged tissues may contribute marker TBX1 in VAT of both active and inactive patients. to increasing succinate levels in CD patients. Further studies An additional experiment treating ASCs isolated from active will be required to understand the origin of succinate levels in CD patients with succinate resulted in a significant increase patients with CD. of TBX1 [0.81 ± 0.15 vs 3.6 ± 0.42, p = 0.0116, data not Chouchani and colleagues recently demonstrated that shown], indicating that succinate could switch white adipo- UCP1+ adipocytes modulate liver extracellular succinate cyte progenitors into beige adipocyte progenitors. levels, and they elegantly showed that UCP1 facilitates the By contrast, when we repeated the analysis in SAT, with clearance and catabolism of circulating succinate by brown the exception of UCP1 expression only COX7A1 was sig- and beige AT.15 We propose that, similarly, beige adipocytes nificantly higher in patients with active disease and a trend associated with CD might contribute to temper tissue fibrosis for lower gene expression was found in patients with inactive and disease pathogenesis. Fibrosis is a consequence of local disease [Figure 4F]. chronic inflammation,55 and strategies to promote browning Abdominal thermal imaging revealed an evident increase of white AT to reverse fibrosis and chronic inflammation in the intestinal area temperature in patients with active have been proposed in other inflammation-related diseases CD [Figure 5A]. Indeed, this analysis revealed an average including liver steatosis56 and may have also beneficial effects peak temperature of 35.76 ± 0.49°C for patients with active in CD. CD, 34.23 ± 0.55°C for patients with inactive disease and We demonstrate that the AT of patients with active disease 33.88 ± 0.78°C for healthy controls [Figure 5B]. Notably, the has a beige phenotype defined by an increase in the expres- minimum temperature for patients with active and inactive sion of UCP1, which is typically absent in white AT. Notably, disease and healthy subjects was 33.68 ± 0.77, 30.4 ± 0.60 UCP1 expression in ASCs correlates with circulating suc- and 30.09 ± 0.93°C, respectively; and the average tempera- cinate levels, and it is possible that succinate regulates UCP1 ture was 34.66 ± 0.49, 32.15 ± 0.61 and 30.09 ± 1.10°C, in preadipocytes to prevent chronic inflammation and fi- respectively [Figure 5B]. We found no significant differences brosis, perhaps mediated by SUCNR1 signalling. However, between the three groups in relation to AT indices analysed further investigations are needed to confirm this. Intriguingly, by bio-impedance [fat-free mass index, fat mass index or total succinate treatment increased the expression of the beige cell VAT] [Supplementary Table 3]. As might be expected,44,45 a progenitor marker, TBX1, in ASCs isolated from CD subjects. significant decrease in skeletal muscle mass and phase angle Our data point to other functions of succinate apart from was found in patients with active disease [Supplementary controlling the potential of beige adipogenesis of ASCs;5 suc- Table 3]. cinate may also act directly in the progenitor adipocyte cell, switching white progenitor cells into beige progenitor cells, and thus limiting white adipogenesis from the beginning. 4. Discussion Creeping fat is an extremely complex environment, and re- In the present study we report, for the first time to our know- cent studies have shown that inflammation-induced barrier dys- ledge, a browning phenotype in the creeping fat and other AT function triggers bacterial translocation.1,57 We found that E. coli depots of patients with active CD. Creeping fat is a recog- co-cultured with ASCs isolated from patients with CD increased nized source of adipocyte-produced soluble factors including UCP1 expression together with RAB5A gene expression, but inflammatory and anti-inflammatory cytokines;46,47 however, only in ASCs from patients with active disease. This may indi- the cell types involved in anti-inflammatory responses are cate an activation of the phagocytosing ability of ASCs, which Adipose Tissue Browning in Crohn’s Disease 1579 B 1. Negative control A Control-ASC 200 Control-ASC + E.coli * Relative gene expression 150 100 50 4 3 2 1 50 µm 0 UCP1 TNFA RAB5A 2. CD-ASC without bacteria aCD-ASC 500 * aCD-ASC + E.coli 400 Relative gene expression 300 200 100 6 50 µm * * 4 3. CD-ASC with bacteria 2 0 UCP1 TNFA RAB5A 200 * iCD-ASC 150 Relative gene expression iCD-ASC + E.coli 50 µm 100 50 In red, UCP1; in blue, nucleus of ASC 4 UCP1 IF quantification 3 * C 2 $ 1 30 0 * UCP1 TNFA RAB5A Area, in % (A.U.) 20 10 * 0 1 2 3 Figure 3. E. coli promotes browning in adipose stem cells isolated from creeping fat. [A] Relative gene expression analysis of the browning marker UCP1, the inflammatory marker TNFA and the phagocytic marker RAB5A in adipose stem cells [ASCs] isolated from visceral adipose tissue [VAT] of patients with active or inactive Crohn’s disease [CD] or from healthy controls, co-cultured or not for 3 h with E. coli. Bars in graphs represent mean ± SEM and significant differences vs cells without E. coli [*p < 0.05] [n = 3 per group]. [B] Immunofluorescence of UCP1 [red label] in ASCs isolated from patients with active CD co-cultured or not with E. coli [3 h]. DAPI staining of nuclei in blue. *p < 0.05 vs control [n = 4 per group]. [C] UCP1 immunofluorescence quantification [% of fluorescence area, mean values ± SEM] [n = 4 per group]. 1580 D. Monfort-Ferré et al. A B Control Active CD Inactive CD $ Control Active CD Inactive CD KDa 4 2.5 PRDM16/β-actin (A.U.) 150 UCP1/β-actin (A.U.) *** PRDM16 140 3 2.0 UCP1-Relative gene expression 1.5 100 $ 2 SAT UCP1 33 1.0 * 1 0.5 50 β-actin 42 0 0.0 20 8 4 15 PRDM16/β-actin (A.U.) PRDM16 140 UCP1/β-actin (A.U.) 6 3 * 10 UCP1 33 5 VAT 4 2 0 β-actin 42 2 1 SAT VAT SAT VAT SAT VAT 0 0 Control Active CD Inactive CD C D Control Active CD Inactive CD 30 ** 25 UCP1-IHC quantification 20 * SAT * Area, in %(a.u.) 15 * 10 5 1.0 0.8 0.6 0.4 VAT 0.2 0.0 SAT VAT SAT VAT SAT VAT Control Active CD Inactive CD E 150 *** 100 VAT-Control Relative gene expression * VAT-Active CD 50 VAT-Inactive CD 6 $ $$ 4 * * ** * 2 0 UCP1 PRDM16 TMEM26 CPTB1 ELOVL3 ADRB3 TBX1 FOXC2 COX7A1 PPARGC1 PPARGC1B F 60 * 40 SAT-Control Relative gene expression SAT-Active CD 20 SAT-Inactive CD 5 * 4 3 2 1 0 UCP1 PRDM16 TMEM26 CPTB1 ELOVL3 ADRB3 TBX1 FOXC2 COX7A1 PPARGC1 PPARGC1B Figure 4. White-to-beige adipose tissue conversion in the subcutaneous and visceral fat depots of patients with Crohn’s disease. [A] Relative UCP1 gene expression in subcutaneous [SAT] and visceral [VAT] adipose tissue samples of patients with active or inactive disease and in healthy individuals. [B] Western blotting of PRDM16 and UCP1; β actin was used as a loading control. Image and densitometry analysis [a.u., arbitrary units] [n = 3 per group]. All data are shown as mean ± SEM; *p < 0.05 [two-tailed unpaired t-test]. [C] Representative immunohistochemical staining of UCP1 in SAT and VAT tissue sections of the different groups and UCP1 quantification [area in percentage of staining] [n = 3–6 per group]. All data are shown as mean ± SEM; *p < 0.05 [two-tailed unpaired t-test]. Relative gene expression of several browning markers in [D] VAT and [E] SAT in active [n = 9] and inactive [n = 7] disease and in healthy individuals [n = 6]. Bars in graphs represent mean ± SEM. *p < 0.05 vs control; $p < 0.05 and $$p < 0.01 as indicated in the figure. Adipose Tissue Browning in Crohn’s Disease 1581 A Control Active CD Inactive CD Sp1 27,3 °C °C 35,0 Bx1 Max 35,7 °C °C 35,0 Bx1 Max 35,0 °C °C 35,0 Bx1 Max 34,3 °C Min 32,5 °C Min 30,7 °C Min 30,4 °C Average 34,2 °C Average 32,2 °C Average 31,7 °C Sp1 31,9 °C Sp1 31,4 °C Bx1 Bx1 Bx1 28,9 28,9 28,9 Sp1 Sp1 Sp1 N41°7'31,36'' N41°7'29,39'' N41°7'30,83'' E1°14'15,67'' E1°14'16,27'' E1°14'15,92'' B 40 $ $$ * $$ Control ** Temperature (°C) 35 ** Active CD Inactive CD 30 25 Temperature Max. Min. Average Figure 5. Abdominal temperature is elevated in patients with active Crohn’s disease. [A] Representative abdominal thermal images of a healthy female, a female with inactive Crohn’s disease [CD] and a female with active CD, matched by age and body mass index. [B] Maximum, minimum and average temperature of intestinal areas were obtained by selecting the area of interest with the rectangle in the FLIR tools program. The average temperature is significantly higher in active CD with respect to healthy controls and is significantly lower in inactive CD with respect to active CD. Bars in graphs represent mean ± SEM. *p = 0.001; **p < 0.0001 vs control; $p = 0.001 and $$p < 0.0001 as indicated in the figure. may have a key role in this process. Our findings also indicate Funding that bacterial translocation might participate in ASC differenti- This study was supported by grants from the Spanish ation into beige adipocytes. These experiments have been per- Ministry of Science and Innovation [PI18/00037 (Instituto formed in undifferentiated ASCs instead of adipocytes, which de Salud Carlos III, ISCIII) to C.S.; PI17/01503 and may be considered a limitation, but we have provided evidence PI20/00338 (ISCIII) to J.V. and RTI2018-093919 to S.F.-V.], that the changes we have observed in the ASCs [pre-adipocytes] co-financed by the European Regional Development Fund are subsequently transferred with an increase overall in protein [ERDF] and a European Crohn’s and Colitis Organization and gene expression of UCP1 in VAT of active CD patients. grant to C.S. The Spanish Biomedical Research Center in This study has some limitations that warrant discussion. It is Diabetes and Associated Metabolic Disorders [CIBERDEM] an observational study, with a limited number of cases, and it [CB07708/0012] is an initiative of the Instituto de Salud would be necessary to monitor the evolution of the patients across Carlos ISCIII acknowledges support from the ‘Ramón time to evaluate changes in beige adipose tissue along the clinical y Cajal’ programme from the Ministerio de Educación course and to disentangle correlation from causality regarding y Ciencia [RYC2013-13186], co-financed by the ERDF. creeping fat and CD. Larger prospective studies will therefore S.F.-V. acknowledge support from the ‘Miguel Servet’ ten- be needed to decipher the clinical relevance of these findings. A ure track programme [CP10/00438, CPII16/0008] from limitation in the use of infrared thermography is that it may be the Fondo de Investigación Sanitaria, co-financed by the affected by several conditions; however, we endeavoured to con- ERDF. D.M.-F. acknowledges support from PERIS-PFI-Salut trol several environmental, technical and individual factors. For SLT01720000021. example, patients were fasted, and room temperature, room size and distance for imaging were all controlled. Also, patients came for imaging on the same day and were matched by sex, age and Conflict of Interest BMI. A final limitation is the relatively small number of subjects included in the study, but we considered this a pilot study to be The authors have no conflicts of interest to declare. validated in future larger cohorts. In conclusion, we provide evidence suggesting that both bac- terial translocation into the creeping fat and the metabolite suc- Acknowledgments cinate act as activators of white AT browning in ASCs from We wish to particularly acknowledge the patients and the patients with CD. This hitherto unrecognized feature of creeping BioBank IISPV [PT17/0015/0029] integrated in the Spanish fat in particular and of AT in general in CD might be exploited to National Biobanks Network for its collaboration. We thank Dr develop new therapeutic or diagnostic strategies in these patients. Kenneth McCreath for helpful comments on the manuscript. 1582 D. Monfort-Ferré et al. Author Contributions 12. Nadjsombati MS, McGinty JW, Lyons-Cohen MR, et al. Detection of succinate by intestinal tuft cells triggers a type 2 innate immune D.M.-F., A.B.-T., C.N.-R. and C.S. performed the experi- circuit. Immunity 2018;49:33–41.e7. ments. A.C., M.M., M.Marti, B.E., M.B. and E.E. and car- 13. Peruzzotti-Jametti L, Bernstock JD, Vicario N, et al. Macrophage- ried out part of the study population selection and human derived extracellular succinate licenses neural stem cells to suppress sample processing. J.S. performed proteomics data analysis. chronic neuroinflammation. Cell Stem Cell 2018;22:355–68.e13. C.S., A.M., J.V. and S.F.-V. contributed to the discussion and 14. Mills EL, Pierce KA, Jedrychowski MP, et al. Accumulation of suc- reviewed the manuscript. C.S. and S.F.-V. conceived the study, cinate controls activation of adipose tissue thermogenesis. Nature discussed data and wrote the manuscript. S.F-V. and C.S. are 2018;560:102–6. the guarantors of this work. 15. Mills EL, Harmon C, Jedrychowski MP, et al. UCP1 governs liver extracellular succinate and inflammatory pathogenesis. Nat Metab 2021;3:604–17. 16. Liu K, Lin L, Li Q, et al. Scd1 controls de novo beige fat biogenesis Ethics Approval through succinate-dependent regulation of mitochondrial complex This study was approved by the ethics committee of II. Proc Natl Acad Sci U S A 2020;117:2462–72. the University Hospital Joan XXIII [CEIM 080/2021, 17. Han SJ, Glatman Zaretsky A, Andrade-Oliveira V, et al. 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