Serena et al. Clinical Epigenetics (2020) 12:53 https://doi.org/10.1186/s13148-020-00843-3 RESEARCH Open Access Adipose stem cells from patients with Crohn’s disease show a distinctive DNA methylation pattern Carolina Serena1,2, Monica Millan3,4, Miriam Ejarque1,2, Alfonso Saera-Vila1, Elsa Maymó-Masip1,2, Catalina Núñez-Roa1,2, Diandra Monfort-Ferré1, Margarida Terrón-Puig1,2, Michelle Bautista5, Margarita Menacho5, Marc Martí6, Eloy Espin6, Joan Vendrell1,2,7* and Sonia Fernández-Veledo1,2* Abstract Background: Crohn’s disease (CD) is characterized by persistent inflammation and ulceration of the small or large bowel, and expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-derived stem cells (hASCs) from CF of patients with CD exhibit dysfunctional phenotypes, including a pro-inflammatory profile, high phagocytic capacity, and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission and are found in all adipose depots explored including subcutaneous fat. We hypothesized that changes in hASCs are a consequence of epigenetic modifications. Methods: We applied epigenome-wide profiling with a methylation array (Illumina EPIC/850k array) and gene expression analysis to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat of patients with CD and healthy controls (n = 7 and 5, respectively; cohort I). Differentially methylated positions (p value cutoff < 1 × 10−4 and ten or more DMPs per gene) and regions (inclusion threshold 0.2, p value cutoff < 1 × 10−2 and more than 2 DMRs per gene) were identified using dmpfinder and Bumphunter (minfi), respectively. Changes in the expression of differentially methylated genes in hASCs were validated in a second cohort (n = 10/10 inactive and active CD and 10 controls; including patients from cohort I) and also in peripheral blood mononuclear cells (PBMCs) of patients with active/inactive CD and of healthy controls (cohort III; n = 30 independent subjects). Results: We found a distinct DNA methylation landscape in hASCs from patients with CD, leading to changes in the expression of differentially methylated genes involved in immune response, metabolic, cell differentiation, and development processes. Notably, the expression of several of these genes in hASCs and PBMCs such as tumor necrosis factor alpha (TNFA) and PR domain zinc finger protein 16 (PRDM16) were not restored to normal (healthy) levels after disease remission. Conclusions: hASCs of patients with CD exhibit a unique DNA methylation and gene expression profile, but the expression of several genes are only partially restored in patients with inactive CD, both in hASCs and PBMCs. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies. Keywords: Methylome, Inflammatory bowel disease, Adipose tissue, Epigenetics, Gene expression * Correspondence: [email protected]; [email protected] 1Institut d´Investigació Sanitària Pere Virgili, Hospital Universitari Joan XXIII, Dr Mallafré Guasch, 4, 43007 Tarragona, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Serena et al. Clinical Epigenetics (2020) 12:53 Page 2 of 15 Background and third, we assessed the reproducibility of the changes The prevalence of inflammatory bowel disease (IBD), in- in gene expression of candidates genes in an more easily cluding Crohn’s disease (CD) and ulcerative colitis, is in- accessible sample, by testing for a specific blood signature. creasing, especially in western countries, and it is likely to represent an important social and economic burden Results in the coming years [1, 2]. CD is characterized by per- Methylation signature of adipose stem cells from sistent inflammation and ulcerations at the small or subcutaneous fat in patients with Crohn’s large bowel, provoking chronic low-grade systemic in- We first assessed genome-wide patterns of DNA methy- flammation and an undulating course of activity, with lation in hASCs isolated from subcutaneous fat depots relapsing cycles occurring after periods of remission. of patients with CD and compared them with those from Adipose tissue (AT) is believed to play an active role in hASCs isolated from healthy controls (n = 7 patients the pathogenesis of CD, as the expansion of mesenteric with CD and 5 healthy controls; cohort I). No differ- fat attached to the inflamed segments of intestine, also ences in overall mean DNA methylation (calculated by known as “creeping fat,” is a hallmark of the disease that averaging across all probes on the Illumina EPIC/850k seems to be directly related to disease activity [3–6]. In array) were observed between the two groups (p = 0.33; fact, it has recently been demonstrated that inclusion of Student’s t test) (data not shown), indicating that CD is the mesentery in ileocolic resection reduces the rate of not associated with global changes to DNA methylation surgical recurrence [7]. in this cell type. By contrast, DNA methylation at indi- AT is now recognized as a highly active endocrine vidual CpG sites showed considerable variability between organ that controls metabolic homeostasis. Emerging CD and controls. Principal component analysis (PCA) of evidence points to the immune system as a major con- the first two principal components (PC1 and PC2) of the tributor to AT homeostasis and function [8]. Indeed, AT methylation data revealed a difference between hASCs contributes importantly to low-grade systemic inflamma- of CD and those of controls (Fig. 1a) with PC1 explain- tion in obesity and its comorbidities [9–11]. AT contains ing 20.56% of the variance. We also performed a correl- an abundant population of multipotent human adipose- ation analysis between the confounding variables, derived stem cells (hASCs) that differentiate into several hospital, sex, age, and body mass index (BMI) and the specialized cells, including adipocytes and endothelial principal components (Fig. 1B; data is represented by a cells, and play key roles in immune response [12, 13]. heat map). No correlation was found between the six For example, hASCs administered to the circulation principal components and hospital, sex, age, or BMI, have the ability to home to inflamed/injured tissues [14, suggesting that these confounding variables did not 15]. Of note, some metabolic stress-induced conditions significantly affect the methylome pattern. Notably, we including CD alter the anti-inflammatory properties of found a significant correlation between the presence or these cells [16–18]. Remarkably, the altered properties of absence of the disease and PC1 (Fig. 1b). hASCs persist even when patients with CD are in remis- It is now recognized that, in addition to the expected sion, in all AT depots explored including subcutaneous binary behavior of CpG methylation (either hypo- or fat [18]. The constancy of this dysfunctional phenotype hypermethylated), some cytosines show intermediate, yet might reflect changes at the epigenetic level, which ul- low, levels of methylation [22]. Thus, we explored poten- timately hamper the resolution of the inflammatory tial differences in the segmented methylomes as fully events in the sinus of the affected mesenteric AT. In methylated regions (FMRs; > 50% methylated CpGs), support of this proposition, recent DNA methylation stud- low-methylated regions (LMRs; 3.9–50%), and unmethy- ies in peripheral blood mononuclear cells (PBMCs) revealed lated regions (UMRs; 1–3.9%), as described [22–26]. The that patients with CD have a distinct DNA methylome FMR class comprised over 50% of the methylome in all linked to the expression of differentially methylated genes samples (Fig. 1c), and the global percentage of methyla- associated with immune response and inflammation [19– tion was stable between control- and CD-hASCs. Never- 21]. We hypothesized that hASCs from patients with CD theless, there was a trend for greater methylation in the are conditioned by a chronic inflammatory milieu, which LMR region in hASCs isolated from patients with CD may alter their DNA methylome and influence their anti- relative to those from healthy controls. inflammatory and regenerative capacity. To address this issue, we designed a three-step study: Differentially methylated positions in adipose stem cells first, we evaluated whether