Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis
Rose L. Szabady, … , Randall J. Mrsny, Beth A. McCormick
Rose L. Szabady, … , Randall J. Mrsny, Beth A. McCormick
Published August 13, 2018
Citation Information: J Clin Invest. 2018;128(9):4044-4056. https://doi.org/10.1172/JCI96817.
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Research Article Gastroenterology

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis

Abstract

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine–type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine.

Authors

Rose L. Szabady, Christopher Louissaint, Anneke Lubben, Bailu Xie, Shaun Reeksting, Christine Tuohy, Zachary Demma, Sage E. Foley, Christina S. Faherty, Alejandro Llanos-Chea, Andrew J. Olive, Randall J. Mrsny, Beth A. McCormick

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Figure 3

P-gp–dependent apical secretion of ECs inhibits neutrophil migration.

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P-gp–dependent apical secretion of ECs inhibits neutrophil migration. (A...
(A) Commercially available ECs and related compounds were tested in the 96-well migration assay, with AEA, OEA, and α-LEA significantly suppressing neutrophil movement. Compounds were used at the highest concentration at which they were soluble in PBS. Data are shown as mean ± SEM of at least 3 independent experiments. *P < 0.05; **P < 0.01, 1-way ANOVA. (B) Confluent 2D human intestinal organoid cultures were used to examine apical secretion of AEA over a 6-hour time course with and without 50 μM verapamil (apical untreated, apical with verapamil), with levels being reported as average of triplicate measurements (± SD) normalized to time 0 values. Increasing apical compartment levels of AEA over time were suppressed by the presence of 50 μM verapamil. Data are shown as mean ± SD from 3 independent experiments with 12–24 technical replicates in each experiment. *P < 0.01; **P < 0.001, unpaired t test. (C) The levels of AEA present in the basal compartment of confluent 2D human intestinal organoid cultures remained relatively constant over this same 6-hour time course, but were reduced in cultures treated with 50 μM verapamil. Relative changes in the basal compartment AEA levels were measured at 0 and 6 hours, with (white bars) and without (black bars) 50 μM verapamil addition. AEA levels are reported as normalized to time 0 values. Data are shown as mean ± SD from 3 independent experiments with 12–24 technical replicates in each experiment. *P < 0.01, unpaired t test.