Genome-wide DNA methylome and transcriptome profiling reveals key genes involved in the dysregulation of adipose-stem cells in Crohn's disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is characterised by the expansion of mesenteric adipose tissue (MAT), named creeping fat (CF), which seems to be directly related to disease activity. Adipose-stem cells (ASCs) isolated from the CF of patients with CD are extremely pro-inflammatory, which persists during disease remission. We hypothesised that the dysfunctional ASCs in CD accumulate epigenetic modifications triggered by the inflammatory environment that could serve as molecular markers. METHODS: Genome-wide DNA methylome and transcriptome profiling were performed in ASCs isolated from MAT adipose-tissue biopsies of patients with active and inactive disease and from non-Crohn's disease patients (non-CD). A validation cohort was used to test the main candidate genes via qPCR in other fat depots and immune cells. RESULTS: We found differences in DNA-methylation and gene expression between ASCs isolated from patients with CD and from non-CD subjects, but we found no differences related to disease activity. Pathway enrichment analysis revealed that oxidative stress and immune response were significantly enriched in active CD and integration analysis identified MAB21L2, a cell fate-determining gene, as the most affected gene in CD. Validation analysis confirmed the elevated gene expression of MAB21L2 in MAT and in adipose tissue macrophages in active CD. We also found a strong association between expression of the calcium channel subunit gene CACNA1H and disease remission, as CACNA1H expression was higher in ASCs and MAT from patients with inactive CD, and correlates negatively with C-reactive protein in peripheral blood mononuclear cells. CONCLUSION: We identified a potential gene signature of CD in ASCs obtained from MAT. Integration analysis highlighted two novel genes demonstrating a negative correlation between promoter DNA methylation and transcription: one linked to ASCs in CD (MAB21L2) and the other (CACNA1H) related to disease remission.

Smoking Suppresses the Therapeutic Potential of Adipose Stem Cells in Crohn's Disease Patients through Epigenetic Changes.

Patients with Crohn's disease (CD) who smoke are known to have a worse prognosis than never-smokers and a higher risk for post-surgical recurrence, whereas patients who quit smoking after surgery have significantly lower post-operative recurrence. The hypothesis was that smoking induces epigenetic changes that impair the capacity of adipose stem cells (ASCs) to suppress the immune system. It was also questioned whether this impairment remains in ex-smokers with CD. ASCs were isolated from non-smokers, smokers and ex-smokers with CD and their interactions with immune cells were studied. The ASCs from both smokers and ex-smokers promoted macrophage polarization to an M1 pro-inflammatory phenotype, were not able to inhibit T- and B-cell proliferation in vitro and enhanced the gene and protein expression of inflammatory markers including interleukin-1b. Genome-wide epigenetic analysis using two different bioinformatic approaches revealed significant changes in the methylation patterns of genes that are critical for wound healing, immune and metabolic response and p53-mediated DNA damage response in ASCs from smokers and ex-smokers with CD. In conclusion, cigarette smoking induces a pro-inflammatory epigenetic signature in ASCs that likely compromises their therapeutic potential.

Anti-TNF Therapies Suppress Adipose Tissue Inflammation in Crohn's Disease.

Anti-TNF biologics have been shown to markedly improve the quality of life for patients with Crohn's disease (CD), yet one-third of patients fail to benefit from this treatment. Patients with CD develop a characteristic wrapping of visceral adipose tissue (VAT) in the inflamed intestinal area, termed creeping fat, and it is known that adipose tissue expansion influences the efficacy of anti-TNF drugs. We questioned whether anti-TNF therapies impact the creeping fat in CD, which might affect the outcome of the disease. Adipose tissue biopsies were obtained from a cohort of 14 patients with CD that received anti-TNF drugs and from 29 non-anti-TNF-treated patients (control group) matched by sex, age, and body mass index undergoing surgical interventions for symptomatic complications. We found that anti-TNF therapies restored adipose tissue morphology and suppressed immune cell infiltration in the creeping fat. Additionally, anti-TNF treatments appeared to markedly improve the pro-inflammatory phenotype of adipose-tissue macrophages and adipose-tissue-derived stem cells. Our study provides evidence that anti-TNF medications influence immune cells and progenitor cells in the creeping of patients with CD, suppressing inflammation. We propose that perilesional VAT should be considered when administering anti-TNF therapy in patients with CD.

The Gut Microbiota Metabolite Succinate Promotes Adipose Tissue Browning in Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease [CD] is associated with complex microbe-host interactions, involving changes in microbial communities, 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 receptor 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 expression, 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 development of therapeutic or diagnostic interventions.

Microbial Signature in Adipose Tissue of Crohn's Disease Patients.

Crohn's disease (CD) is characterized by compromised immune tolerance to the intestinal commensal microbiota, intestinal barrier inflammation, and hyperplasia of creeping fat (CF) and mesenteric adipose tissue (AT), which seems to be directly related to disease activity. Gut microbiota dysbiosis might be a determining factor in CD etiology, manifesting as a low microbial diversity and a high abundance of potentially pathogenic bacteria. We tested the hypothesis that CF is a reservoir of bacteria through 16S-rRNA sequencing of several AT depots of patients with active and inactive disease and controls. We found a microbiome signature within CF and mesenteric AT from patients, but not in subcutaneous fat. We failed to detect bacterial DNA in any fat depot of controls. Proteobacteria was the most abundant phylum in both CF and mesenteric AT, and positively correlated with fecal calprotectin/C-reactive protein. Notably, the clinical status of patients seemed to be related to the microbiome signature, as those with the inactive disease showed a reduction in the abundance of pathogenic bacteria. Predictive functional profiling revealed many metabolic pathways including lipopolysaccharide biosynthesis and sulfur metabolism overrepresented in active CD relative to that in inactive CD. Our findings demonstrate that microbiota dysbiosis associated with CD pathophysiology is reflected in AT and might contribute to disease severity.

Adipose stem cells from patients with Crohn's disease show a distinctive DNA methylation pattern.

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. Human adipose-stem cells isolated from subcutaneous fat of patients with Crohn's disease exhibit an altered DNA methylation pattern and gene expression profile compared with those isolated from healthy individuals, with immune system, cell differentiation, metabolic and development processes identified as the main pathways affected. Interestingly, the gene expression of several genes involved in these pathways is only partially restored to control levels in patients with inactive Crohn's disease, both in human adipose-stem cells and peripheral blood mononuclear cells. Understanding how Crohn's disease shapes the functionality of human adipose-stem cells is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.