Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis
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
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
View: Text | PDF
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

×

Figure 2

Epithelial cells secrete P-gp–dependent ECs that inhibit neutrophil migration.

Options: View larger image (or click on image) Download as PowerPoint
Epithelial cells secrete P-gp–dependent ECs that inhibit neutrophil migr...
(A) Supernatants from T84 epithelial monolayers were enriched for lipids and tested for the ability to inhibit HxA3-induced migration in a 96-well modified Boyden chamber assay. In order to compare across experiments with different donors, migration values within individual experiments were normalized to enriched HxA3 with vehicle treatment. For A–C, data are shown as mean ± SEM of 3 independent experiments. *P < 0.05: ***P < 0.01, 1-way ANOVA. (B) Performed as in A, with supernatants from cell lines expressing different shRNA constructs to knock down P-gp expression (B4-MDR1 and B5-MDR1). (C) Performed as in A, but prior to use in the migration assays, enriched T84 supernatants were pretreated with FAAH or MAGL at 37°C for 30 minutes. (D) Lipid-extracted T84 supernatants from control or 2 separate MDR1-knockdown cell lines (B4 and B5) were subjected to electrospray ionization MS. AEA-d8 was included in each sample to allow quantitative comparisons between samples and was used to calculate relative units of each EC. Individual ECs were normalized to the relative units in the scrambled control condition.