Macrophage-derived EDA-A2 inhibits intestinal stem cells by targeting miR-494/EDA2R/β-catenin signaling in mice

The mucosa microenvironment is critical for intestinal stem cell self-renewal and reconstruction of the epithelial barrier in inflammatory bowel disease (IBD), where the mechanisms underlying cross-talk between intestinal crypts and the microenvironment remain unclear. Here, we firstly identified miR-494-3p as an important protector in colitis. miR-494-3p levels were decreased and negatively correlated with the severity in human IBD samples, as well as in colitis mice. In colitis crypts, a notable cytokine–cytokine receptor, miR-494-3p-targeted EDA2R and the ligand EDA-A2, suppressed colonic stemness and epithelial repair by inhibiting β-catenin/c-Myc. In differentiated IECs, miR-494-3p inhibits macrophage recruitment, M1 activation and EDA-A2 secretion by targeting IKKβ/NF-κB in colitis. A miR-494-3p agomir system notably ameliorated the severity of colonic colitis in vivo. Collectively, our findings uncover a miR-494-3p-mediated cross-talk mechanism by which macrophage-induced intestinal stem cell impairment aggravates intestinal inflammation.


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October 2018

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Colon tissues and sera were collected from IBD patients who had been pathologically confirmed to have active UC (A-UC) or CD (A-CD). Colon tissues were taken at the time of endoscopy and uninflamed tissues from the respective patient were used as controls. Sera from healthy volunteers and active IBD patients were collected in the course of blood collection for clinical examination. The age at presentation varied from 20 to 67 years. The gender distribution was about 1:1. The detail characteristics (age, gender, current therapy, disease extent (UC), disease location (CD), and CRP) were included in the " Table  S4" section of the supplementary materials.
All of patients were recruited from the Shanghai Tenth People's Hospital of Tongji University (Shanghai, China).Colon tissues were taken at the time of endoscopy and uninflamed tissues from the respective patient were used as controls. Sera from healthy volunteers and active IBD patients were collected in the course of blood collection for clinical examination. No bias was identified. Before the study, we obtained written informed consent from all participants.
The use of human specimens was approved by the Ethics Review Board of the Shanghai Tenth People's Hospital (Tongji University).
Lamina Propria Lymphocytes (LPL) For isolation of LPL, the whole colon was removed, cut into fragments of about 1 cm in length, and rinsed with HBSS buffer. The rinsed colon fragments then were gently shaken (250 rpm) for 30 min at 37°C in HBSS buffer supplemented with 30 mM EDTA (sigma, Darmstadt, Germany), 1 mM DTT (sigma), and 5% (vol/vol) FBS. After sedimentation by standing, the supernatant was discarded and the remaining colon tissues were further cut into smaller pieces and incubated with digestive solution [RPMI-1640 medium (Gibco) supplemented with 200 U/mL collagenase VIII (sigma), 150 "g/mL DNAase I (sigma), 5% (vol/vol) FBS, and 1% (vol/vol) penicillin and streptomycin] for 1 hour at 37°C. After digestion, the supernatant was centrifuged at 450xg for 5 min and Percoll (40%/80%; GE Healthcare) was used to isolate LPLs. The LPLs then were subjected to flow cytometry.
splenic Immune Cells For isolation of immune cells from the spleen, spleen was crushed and passed through a 40-"m filter to obtain a single-cell