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Mesenteric Adipose-derived Stromal Cells From Crohn’s Disease Patients Induce Protective Effects in Colonic Epithelial Cells and Mice With Colitis

2018; Elsevier BV; Volume: 6; Issue: 1 Linguagem: Inglês

10.1016/j.jcmgh.2018.02.001

2352-345X

Jill M. Hoffman, Aristea Sideri, Jonathan J. Ruiz, Dimitris Stavrakis, David Q. Shih, Jerrold R. Turner, Charalabos Pothoulakis, Ιορδάνης Καραγιαννίδης,

Infant Nutrition and Health

Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media–treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation. Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media–treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation. See editorial on page 113. See editorial on page 113. SummaryExtracellular mediators from mesenteric adipose-derived stromal cells (ADSCs) promote colonocyte proliferation and the resolution of inflammation in a disease-dependent manner. ADSC-derived mediators such as lactoferrin may provide therapeutic potential to promote remission from intestinal inflammation in Crohn's disease patients. Extracellular mediators from mesenteric adipose-derived stromal cells (ADSCs) promote colonocyte proliferation and the resolution of inflammation in a disease-dependent manner. ADSC-derived mediators such as lactoferrin may provide therapeutic potential to promote remission from intestinal inflammation in Crohn's disease patients. Crohn's disease (CD) and ulcerative colitis, collectively known as inflammatory bowel disease (IBD), are chronic relapsing inflammatory disorders of the gastrointestinal tract. A specific feature of CD is white adipose tissue hyperplasia that likely develops from mesenteric depots residing in close proximity to the abdominal wall. Mesenteric adipose tissue expansion and wrapping of the involved intestinal segment occur during the onset of inflammation in CD.1Desreumaux P. Ernst O. Geboes K. Gambiez L. Berrebi D. Muller-Alouf H. Hafraoui S. Emilie D. Ectors N. Peuchmaur M. Cortot A. Capron M. Auwerx J. Colombel J.F. 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Adipose-derived stromal/stem cells: a primer.Organogenesis. 2013; 9: 3-10Crossref PubMed Scopus (83) Google Scholar which contribute to the metabolic and immunologic functions of fat by synthesizing and secreting hormones, cytokines, and chemokines.9Fantuzzi G. Adipose tissue, adipokines, and inflammation.J Allergy Clin Immunol. 2005; 115: 911-920Abstract Full Text Full Text PDF PubMed Scopus (1952) Google Scholar The expansion of adipose tissue during obesity promotes the development of a chronic low-grade inflammation and is a significant risk factor for the development of many chronic inflammatory diseases.10Hotamisligil G.S. Erbay E. Nutrient sensing and inflammation in metabolic diseases.Nat Rev Immunol. 2008; 8: 923-934Crossref PubMed Scopus (782) Google Scholar, 11Odegaard J.I. Chawla A. Mechanisms of macrophage activation in obesity-induced insulin resistance.Nature Clinical Practice Endocrinology Metabolism. 2008; 4: 619-626Crossref PubMed Scopus (204) Google Scholar Obesity has a negative impact on the course of CD, associated with increased severity of inflammation12Blain A. Cattan S. Beaugerie L. Carbonnel F. Gendre J.P. Cosnes J. Crohn's disease clinical course and severity in obese patients.Clin Nutr. 2002; 21: 51-57Abstract Full Text PDF PubMed Scopus (172) Google Scholar and an earlier progression to surgery.13Hass D.J. Brensinger C.M. Lewis J.D. Lichtenstein G.R. The impact of increased body mass index on the clinical course of Crohn's disease.Clin Gastroenterol Hepatol. 2006; 4: 482-488Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar Previously, our laboratory demonstrated that mice with trinitrobenzenesulfonic acid (TNBS)–induced colitis display mesenteric fat wrapping of the intestine as well as major inflammatory changes in mesenteric ADSCs including increased expression levels of proinflammatory cytokines and neuropeptide receptors14Karagiannides I. Kokkotou E. Tansky M. Tchkonia T. Giorgadze N. O'Brien M. Leeman S.E. Kirkland J.L. Pothoulakis C. Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (75) Google Scholar, 15Koon H.W. Kim Y.S. Xu H. Kumar A. Zhao D. Karagiannides I. Dobner P.R. Pothoulakis C. Neurotensin induces IL-6 secretion in mouse preadipocytes and adipose tissues during 2,4,6,-trinitrobenzensulphonic acid-induced colitis.Proc Natl Acad Sci U S A. 2009; 106: 8766-8771Crossref PubMed Scopus (56) Google Scholar as well as inflammatory infiltrate, neutrophil and leukocyte migration, and diapedesis.14Karagiannides I. Kokkotou E. Tansky M. Tchkonia T. Giorgadze N. O'Brien M. Leeman S.E. Kirkland J.L. Pothoulakis C. Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (75) Google Scholar Furthermore, we recently showed that CD patient-derived mesenteric ADSCs and whole mesenteric fat tissue exhibit differential patterns of cytokine release compared with control patients.16Sideri A. Bakirtzi K. Shih D.Q. Koon H.W. Fleshner P. Arsenescu R. Arsenescu V. Turner J.R. Karagiannides I. Pothoulakis C. Substance P mediates pro-inflammatory cytokine release form mesenteric adipocytes in inflammatory bowel disease patients.Cell Mol Gastroenterol Hepatol. 2015; 1: 420-432Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Here we hypothesized that mesenteric ADSCs from CD and control patients exhibit differential gene expression profiles and induce disease-dependent responses in intestinal epithelial cells, thus participating in the pathophysiology of CD. To address this hypothesis, we injected conditioned media from cultured ADSCs in mice with experimental colitis in vivo. In addition, we performed microarray analysis of CD and control patient-derived mesenteric ADSCs and assessed the effects of their extracellular mediators on intestinal epithelial cell signaling and gene expression in vitro. Our profiling revealed several differentially regulated molecules, including lactoferrin, which has not been previously identified as a product of adipose tissue. Our results indicate that extracellular mediators released from CD patient-derived mesenteric ADSCs can attenuate the severity of experimental colitis and promote proliferative responses in intestinal epithelial cells, thus supporting a protective role for mesenteric adipose tissue during intestinal inflammation. Mesenteric adipose tissue was resected from male and female patients with severe, active CD presenting for surgery (n = 46) and non-IBD control patients (n = 35) presenting for adenocarcinoma, gynecologic, vascular surgery, or other gastrointestinal complications. Subjects taking thiazolidinediones or steroids were excluded, and patients were fasted for at least 10 hours before surgery. Human studies protocols have been approved by the UCLA Institutional Review Board for Human Research (protocol #11-001527-AM-00003). All participants gave informed consent before taking part. Subjects with malignancies were not excluded because they may constitute an important subpopulation that could yield significant information for our intergroup comparisons. Tissues from Cedars-Sinai Medical Center were obtained after informed consent in accordance with procedures established by the Cedars-Sinai Institutional Review Board (protocol #3358 and #23705). Mesenteric ADSCs were isolated from adipose tissue samples as previously described.16Sideri A. Bakirtzi K. Shih D.Q. Koon H.W. Fleshner P. Arsenescu R. Arsenescu V. Turner J.R. Karagiannides I. Pothoulakis C. Substance P mediates pro-inflammatory cytokine release form mesenteric adipocytes in inflammatory bowel disease patients.Cell Mol Gastroenterol Hepatol. 2015; 1: 420-432Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17Karagiannides I. Tchkonia T. Dobson D.E. Steppan C.M. Cummins P. Chan G. Salvatori K. Hadzopoulou-Cladaras M. Kirkland J.L. Altered expression of C/EBP family members results in decreased adipogenesis with aging.Am J Physiol Regul Integr Comp Physiol. 2001; 280: R1772-R1780Crossref PubMed Google Scholar, 18Sideri A. Stavrakis D. Bowe C. Shih D.Q. Fleshner P. Arsenescu V. Arsenescu R. Turner J.R. Pothoulakis C. Karagiannides I. Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat: potential role of adiponectin receptor 1.Am J Physiol Gastrointest Liver Physiol. 2015; 308: G591-G604Crossref PubMed Scopus (31) Google Scholar Briefly, 2–5 g adipose tissue was minced finely into small pieces by using sterile surgical scissors in 50 mL conical tubes containing 1 mg/mL collagenase (Worthington Biochemical Corporation, Lakewood, NJ) solution in ×1 phosphate-buffered saline (3 mL/g tissue). Minced tissue was vortexed and placed in a shaking water bath (100 rpm, 37°C) for 40 minutes before passing through a sterile 100-μm nylon mesh filter (Thermo Fisher Scientific, Waltham, MA). Homogenates were centrifuged at 1000 rpm for 10 minutes, and the resulting pellet was resuspended in 10 mL erythrocyte lysis buffer (Gibco, Thermo Fisher Scientific) before further centrifugation. The pellet was then resuspended in 10 mL Dulbecco's modified Eagle medium (ATCC, Manassas, VA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 0.1 mmol/L penicillin, and 0.06 mmol/L streptomycin (Gibco, Thermo Fisher Scientific) and plated on 100-mm culture dishes. Isolated cells were cultured as previously described16Sideri A. Bakirtzi K. Shih D.Q. Koon H.W. Fleshner P. Arsenescu R. Arsenescu V. Turner J.R. Karagiannides I. Pothoulakis C. Substance P mediates pro-inflammatory cytokine release form mesenteric adipocytes in inflammatory bowel disease patients.Cell Mol Gastroenterol Hepatol. 2015; 1: 420-432Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17Karagiannides I. Tchkonia T. Dobson D.E. Steppan C.M. Cummins P. Chan G. Salvatori K. Hadzopoulou-Cladaras M. Kirkland J.L. Altered expression of C/EBP family members results in decreased adipogenesis with aging.Am J Physiol Regul Integr Comp Physiol. 2001; 280: R1772-R1780Crossref PubMed Google Scholar, 18Sideri A. Stavrakis D. Bowe C. Shih D.Q. Fleshner P. Arsenescu V. Arsenescu R. Turner J.R. Pothoulakis C. Karagiannides I. Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat: potential role of adiponectin receptor 1.Am J Physiol Gastrointest Liver Physiol. 2015; 308: G591-G604Crossref PubMed Scopus (31) Google Scholar and passaged at least 3 times to ensure removal of macrophages, and conditioned media (1 mL; 24-hour incubation), RNA and protein were collected from confluent cells for analysis. The purity of these cultures was determined to be >90% in studies that compared single cell derived colonies with those of similarly treated skin and lung fibroblasts for their ability to accumulate lipid.19Kirkland J.L. Hollenberg C.H. Gillon W.S. Age, anatomic site, and the replication and differentiation of adipocyte precursors.Am J Physiol. 1990; 258: C206-C210Crossref PubMed Google Scholar The human colonic epithelial cell line NCM460 (INCELL, San Antonio, TX) was incubated at 37°C with 5% CO2 in M3:D culture medium (INCELL) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 10 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Thermo Fisher Scientific). ADSC-derived conditioned media (1 mL) from 6 CD and 6 control patients were added to confluent human colonic NCM460 cells and incubated for 24 hours. RNA was then isolated by using the miRNeasy mini kit (QIAGEN, Redwood City, CA) and subjected to mRNA expression analysis by microarray. In separate experiments, NCM460 cells were treated with human apolactoferrin (50 μg/mL; MyBioSource, Inc, San Diego, CA). Total protein was isolated from cells after treatment with conditioned medium or apolactoferrin by using RIPA buffer and subjected to multiplex protein analysis by using a custom 15-plex phosphoprotein panel or Western blot. The Human LncRNA Microarray V4.0 (Arraystar, Inc, Rockville, MD) was used to examine differential expression of 60,903 distinct transcripts including 20,730 protein-coding mRNAs in total RNA isolated from CD or control patient-derived ADSCs (6 patients/group, selected among those with the highest RNA quality). NCM460 cells were profiled with the GeneChip Human Gene ST Array (Affymetrix, Santa Clara, CA) after treatment with conditioned media from the same CD or control patient-derived ADSCs used for microarray profiling (n = 6 patients/group). The mRNA expression data from both arrays were analyzed by using QIAGEN's Ingenuity Pathway Analysis (IPA; QIAGEN, www.qiagen.com/ingenuity). Male C57BL/6J mice 8–10 weeks old were purchased from the Jackson Laboratory (Sacramento, CA). Mice were housed 4 per cage, maintained on a 12:12 hour light-dark cycle, and given access to food and water ad libitum. To induce colitis, dextran sodium sulfate (DSS; 3.5% or 4% w/v; Affymetrix) was dissolved in drinking water and supplied for 5 or 7 days. Mice received an intracolonic (i.c.) injection (0.1 mL) daily of vehicle (culture medium) or conditioned medium from control or CD patient-derived ADSCs (n = 3 patients/group, selected at random from those profiled above; n = 4 mice/patient) under isoflurane anesthesia. Separate groups of mice received daily injections of 10 mg/mL human apolactoferrin or saline (0.1 mL, i.c.). Mice were weighed and monitored daily and killed on day 5 or 7 by CO2 asphyxiation and cervical dislocation. Severity of colitis was assessed by weight loss and determination of clinical and histologic damage scores as previously described.20Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar, 21Ungaro R. Fukata M. Hsu D. Hernandez Y. Breglio K. Chen A. Xu R. Sotolongo J. Espana C. Zaias J. Elson G. Mayer L. Kosco-Vilbois M. Abreu M.T. A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1167-G1179Crossref PubMed Scopus (146) Google Scholar Briefly, clinical scores were calculated on a scale of 0–3 for degree of weight loss, bleeding, stool consistency, and presence of rectal prolapse on day 5 or 7. Sections of colon (4 μm) fixed in 10% formalin, paraffin-embedded, and stained with H&E were used for histologic scoring by using an AxioImager .Z1 microscope equipped with AxioVision software version 4.6 (Zeiss, Jena, Germany). Scoring was performed by 2 independent investigators blinded to the treatment conditions, and resulting scores were averaged. Scores were assigned on the basis of the degree of crypt damage (0–4), polymorphonuclear neutrophil infiltrate (0–3), erosion (0–3), edema (0–3), and epithelial regeneration (0–3).21Ungaro R. Fukata M. Hsu D. Hernandez Y. Breglio K. Chen A. Xu R. Sotolongo J. Espana C. Zaias J. Elson G. Mayer L. Kosco-Vilbois M. Abreu M.T. A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1167-G1179Crossref PubMed Scopus (146) Google Scholar Adjacent sections were immunostained with a rabbit Ki-67 monoclonal antibody (dilution 1:200; Cell Signaling Technology, Danvers, MA). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. Heat-induced antigen retrieval was performed in 0.01 mol/L citrate buffer, pH = 6 by using a decloaking chamber (Biocare Medical, Pacheco, CA) at 95°C for 25 minutes. The slides were then incubated with the primary antibody for 1 hour at room temperature. Signal was detected by using the rabbit horseradish peroxidase EnVision kit (Dako, Agilent Technologies Inc, Santa Clara, CA) and visualization by the diaminobenzidine reaction. Sections were counterstained with hematoxylin, air-dried, and coverslipped. Digital images were taken by using AxioCam under a ×20 objective lens, and pixel-based quantification of Ki-67 immunoreactivity was performed by using the AutoMeasure module to avoid selection bias or interobserver variation. Images were acquired, tiled, and stitched by using the MosaiX module and data collected from user-defined regions reflecting the entire epithelial surface of each section. All animal protocols were approved by the Institutional Animal Care and Use Committee and the Office of Animal Research Oversight at UCLA. Total RNA was extracted from human ADSCs, NCM460 cells, and mouse colonic tissue using standard Trizol-based protocols, and complementary DNA was generated by reverse-transcriptase (RT) reaction (Promega, Madison, WI). An Applied Biosystems 7500 Fast Real-time Polymerase Chain Reaction (PCR) System was used with Fast Universal PCR Master Mix and validated TaqMan Gene Expression Assays for the detection of interleukin (IL) 1β, CCL2, CXCL1, IL6, IL17A, CCL23, lactoferrin, and vascular endothelial growth factor (VEGF) A (Applied Biosystems, Foster City, CA). Resulting data were calculated by using the 2-[delta][delta]Ct method, and the level of mRNA expression was normalized to Rn18S (Applied Biosystems). Expression of Rn18S was consistent across the treatments and conditions studied. To assess NCM460 cell proliferation we used a label-free real-time cell analysis platform (xCELLigence; ACEA Biosciences, Inc, San Diego, CA) as previously described.22Limame R. Wouters A. Pauwels B. Fransen E. Peeters M. Lardon F. De Wever O. Pauwels P. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.PloS One. 2012; 7: e46536Crossref PubMed Scopus (222) Google Scholar Briefly, 100 μL complete culture medium was added to each well of an E-plate 96 at RT. The E-plate 96 was connected to the system, and background impedance was measured during a period of 24 seconds. Cells were resuspended in complete culture medium and adjusted to 5000 cells/mL, which had been previously determined as the optimal seeding concentration. Resuspended cells were added to the E-plate 96 medium-containing wells. After 30 minutes of incubation at RT the E-plate 96 was placed into the cell culture incubator. Approximately 24 hours after seeding, when the cells were in the log growth phase (Cell Index >0.5), 100 μL ADSC conditioned medium from control and CD patients was added. In a separate experiment, cells were treated with 25 μmol/L NBP2-29332, an Akt 1/2/3 inhibitor (Novus Biologicals, Littleton, CO) or 10 μmol/L CHIR 99021, a glycogen synthase kinase-3 inhibitor (R&D Systems, Inc, Minneapolis, MN). Vehicle controls received non-conditioned medium. Cell proliferation was monitored and recorded every 15 minutes for 72 hours via the incorporated sensor electrode array of the E-plate 96. Electrical impedance was measured with the xCELLigence system software as a dimensionless parameter termed Cell Index, which directly correlates to the proportion of the plate surface occupied by adherent cells.23Atienza J.M. Yu N. Kirstein S.L. Xi B. Wang X. Xu X. Abassi Y.A. Dynamic and label-free cell-based assays using the real-time cell electronic sensing system.Assay and Drug Development Technologies. 2006; 4: 597-607Crossref PubMed Scopus (159) Google Scholar Human mesenteric ADSCs were isolated and plated as described above, and media were collected at the end of the 24-hour period. NCM460 cells were then exposed to ADSC-derived conditioned medium for 24 hours, and total protein was collected by using RIPA buffer (Boston Bioproducts, Boston, MA) with protease and phosphatase inhibitors (Thermo Fisher Scientific). For phosphoprotein detection we used a custom panel that included antibody-loaded magnetic beads against Akt Ser473, GSK3b Ser9, p70SK6 Thr44/Ser424, p65 Ser536, c-Jun Ser63, STAT3 Tyr705, MEK1 Ser47/221, ERK1/2 Thr202/204/Thr185/Tyr187, p38 MAPK Thr180/Tyr182, JNK Thr183/185, PDGFR Tyr754, IRS1 Ser636/639, and IGFR-1 Tyr1131 (Bio-Rad, Hercules, CA), and the final data were obtained and analyzed via the Bio-plex 3D Suspension array system (Bio-Rad). In addition to loading volume, results were normalized for total protein. Western blot analysis was performed in protein lysates from NCM460 cells. Cells were washed with ice-cold phosphate-buffered saline and incubated with radiolabeled immunoprecipitation buffer containing protease inhibitors and sodium orthovanadate (Santa Cruz Biotechnology, Inc, Dallas, TX) for 5 minutes. Cell lysates underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes. Blocking solution contained phosphate-buffered saline, 5% nonfat dry milk, and 0.05% Tween-20, and membranes were probed with primary antibodies for total Akt and phosphorylated Akt and s6p70K (dilution 1:1000; Cell Signaling Inc). The membrane was then blotted with the IRDye 800CW donkey anti-rabbit immunoglobulin G secondary antibody 1:15,000 (LI-COR Biosciences, Lincoln, NE) in blocking buffer for 1 hour. Visualization and quantitation were performed by using the Odyssey CLx Imaging system (LI-COR Biosciences). Enzyme-linked immunosorbent assay for lactoferrin was performed by using the human lactoferrin ELISA kit (LS-F12851; LifeSpan BioSciences, Inc, Seattle, WA), according to the manufacturer's instructions. Statistical analyses were performed by using GraphPad Prism software (Version 5.0a; GraphPad Software, La Jolla, CA). Differences between groups were determined by Mann-Whitney test or one-way analysis of variance, or two-way analysis of variance with Bonferroni or Tukey post-test. A P value <.05 was considered to be statistically significant. Data shown represent the mean ± standard error of the mean. All authors had access to the study data and reviewed and approved the final manuscript. We first determined whether extracellular mediators from CD and control patient-derived mesenteric ADSCs could induce differential responses in the inflamed intestine. To accomplish this, we administered conditioned media from either cultured CD (n = 3) or control patient-derived ADSCs (n = 3) or vehicle via daily i.c. injections in mice with acute DSS (3.5% w/v) colitis for 5 days (Figure 1A). The DSS concentration and time point of these experiments were chosen to allow us to determine whether control or CD patient-derived media can enhance or limit the onset or severity of colitis. Although the degree of weight loss observed was not different among groups of mice with DSS colitis receiving vehicle, control patient-derived or CD patient-derived ADSCs (Figure 1B; n = 12/group), mice with DSS colitis receiving injections of CD patient-derived ADSC media had reduced clinical scores compared with mice receiving control patient media (1.375 ± 0.3239 versus 2.875 ± 0.4407, P < .05; Figure 1C) or vehicle injections (1.375 ± 0.3239 versus 4.125 ± 0.4795, P < .001; Figure 1C). Furthermore, mice receiving injections of CD patient-derived media had reduced histologic scores, compared with vehicle (3.833 ± 0.6009 versus 6.313 ± 0.7130, P < .05; Figure 1D) or control patients (3.833 ± 0.6009 versus 6.313 ± 0.7673, P < .05; Figure 1D). These results suggest that the course of acute DSS colitis is attenuated in the presence of extracellular mediators released from CD patient-derived ADSCs, supporting a protective role for mesenteric adipose tissue in the pathophysiology of CD. In addition to a decrease in the severity of DSS colitis, conditioned media from patient-derived ADSCs altered mRNA expression of proinflammatory cytokines in colonic tissues of mice with acute DSS colitis (Figure 2A–D). Total RNA from full-thickness segments of mouse distal colon was used to measure mRNA expression of proinflammatory cytokines. Media from CD patient-derived ADSCs decreased mRNA expression of IL1β compared with vehicle (P < .01) or control patients (P < .05; Figure 2A). Media from CD patient-derived ADSCs reduced CCL2 and IL6 mRNA expression compared with vehicle (P < .05), whereas CXCL1 mRNA expression was reduced as compared with control p