Human colonic EVs induce murine enteric neuroplasticity via the lncRNA GAS5/miR-23/NMDA NR2B axis
QiQi Zhou, Liuqing Yang, Zachary T. Verne, Benjamin B. Zhang, Jeremy Z. Fields, Amber T. Thacker, G. Nicholas Verne
QiQi Zhou, Liuqing Yang, Zachary T. Verne, Benjamin B. Zhang, Jeremy Z. Fields, Amber T. Thacker, G. Nicholas Verne
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Research Article Gastroenterology

Human colonic EVs induce murine enteric neuroplasticity via the lncRNA GAS5/miR-23/NMDA NR2B axis

Abstract

Postinfectious, diarrhea-predominant, irritable bowel syndrome (PI-IBS-D) is difficult to treat owing to its unknown pathophysiology. Extracellular vesicles (EVs) derived from human colon tissue and long noncoding RNAs (lncRNAs), such as growth arrest–specific 5 (GAS5), may play key roles in the pathophysiology of PI-IBS-D. To determine whether altered colonic EV lncRNA signaling leads to gastrointestinal dysfunction and heightened visceral nociception in patients with PI-IBS-D via the GAS5/miR-23ab/NMDA NR2B axis, we conducted translational studies, including those on (a) the role of colonic EV lncRNAs in patients with PI-IBS-D, human colonoids, and PI-IBS-D tissues; (b) i.p. injection of colonic EVs from patients with PI-IBS-D into Rab27a/b–/– mice (P-EV mice) to investigate whether colonic EVs drive visceral hypersensitivity in vivo via the GAS5/miR-23ab/NMDA NR2B axis; and (c) treatment of mice with oligo-miR-23 precursors and anti-GAS5 Vivo-Morpholinos for GAS5/miR-23ab/NMDA NR2B axis mechanisms. Colonic EVs from patients with PI-IBS-D, but not from control participants, demonstrated reduced miR-23a/b expression caused by enhanced GAS5 expression, which drives increased NR2B expression. Intraperitoneal injection of anti–GAS5-Vivo-Morpholino into P-EV mice increased miR-23 levels and decreased NR2B expression and VMR to CD. EVs are internal messengers that alter gastrointestinal function and increase visceral nociception in patients with PI-IBS-D. Strategies to deliver EVs to modulate GAS5/miR-23ab/NMDA NR2B axis signaling may lead to new and innovative treatments for patients with PI-IBS-D.

Authors

QiQi Zhou, Liuqing Yang, Zachary T. Verne, Benjamin B. Zhang, Jeremy Z. Fields, Amber T. Thacker, G. Nicholas Verne

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Figure 4

Evidence for in vitro endocytosis of colonic EVs and neuronal cells.

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Evidence for in vitro endocytosis of colonic EVs and neuronal cells. (A)...
(A) Coincubation of human neuronal cells (LUHMES, ATCC CRL-2927) with colonic EVs from patients with PI-IBS-D revealed increased NMDA NR2B expression starting at 24 hours and reaching significance at 48 hours. (B and C) Coincubation of human neuronal cells with colonic EVs from patients with PI-IBS-D lead to significantly diminished miR-23a (B) and miR-23b (C) after 24, 48, and 72 hours versus control participants. (D) Coincubation of mouse DRG cells with colonic EVs from patients with PI-IBS-D resulted in significantly increased NMDA NR2B expression at 48 and 72 hours. (E and F) Coincubation of mouse DRGs with colonic EVs from patients with PI-IBS-D lead to decreased miR-23a expression (E) at 24, 48, and 72 hours and decreased miR-23b expression (F) at 48 and 72 hours. (G) IHC showing endocytosis of human colonic EVs (red), neuronal cells (green), and DAPI (blue) after 8 hours of incubation. Scale bars: 10 μm. (H–M) Relative GAS5 (H and I), miR-23a (J and K), and miR-23b (L and M) expression in human colonoids incubated for 24 and 48 hours with colonic EVs from patients with PI-IBS-D or control participants. At 48 hours, GAS5 was significantly increased, and miR-23a and miR-23b were decreased. (N) Endocytosis of human colonic EVs (red) into the colonoids after 48 hours of incubation. Epithelial cell marker (EpCam, green) and EVs labeled with PKH26 (red). Scale bars: 10 μm. *P = 0.05, **P = 0.01 by unpaired t test.